Illumination systems and optical devices for laser beam shaping

ABSTRACT

Various embodiments of a laser illumination system are disclosed. In some examples, a system includes one or more lasers and a beam combiner configured to direct a combined light beam along a path. The system can include a polychroic optical assembly that receives the combined light beam and to output a first beam of the first light of the first wavelength and a second beam of the second light of the second wavelength. The second beam can be offset from the first beam. The polychroic optical assembly can be a prism assembly. The system can include one or more optical elements configured alter a distribution of light of the first beam to output a first output line at a sample plane, and to alter a distribution of light of the second beam to output a second output line at the sample plane.

CROSS-REFERENCE TO RELATED APPLICATIONS

This non-provisional patent application claims priority under 35 U.S.C.119(e) to U.S. Provisional Patent Application No. 63/336,235, filed onApr. 28, 2022, and entitled “ILLUMINATION SYSTEMS AND OPTICAL DEVICESFOR LASER BEAM SHAPING,” which is hereby incorporated by referenceherein in its entirety.

U.S. patent application Ser. No. 17/390,348, filed Jul. 30, 2021, andentitled “LASER SYSTEMS AND OPTICAL DEVICES FOR LASER BEAM SHAPING” ishereby incorporated by reference herein in its entirety. The '348 patentapplication published on May 26, 2022 as U.S. Patent ApplicationPublication No. 2022/0163786, which is hereby incorporated by referencein its entirety. U.S. Pat. No. 10,114,213, issued Oct. 30, 2018, andentitled “LASER SYSTEMS AND OPTICAL DEVICES FOR MANIPULATING LASERBEAMS” is hereby incorporated by reference herein in its entirety.

BACKGROUND Field

This disclosure generally relates to optical illumination systems and todevices for laser beam shaping, such as for optical (e.g., fluorescent,spectroscopic, scatter) analysis of biological samples contained in flowcells. Some implementations relate to, for example, compact, thermallystable multi-laser systems configured to couple to flow cells, opticalfibers, or other target objects and to provide illumination thereto.Aspects of this disclosure also relate generally to optical systems fordirecting light to a sample contained in a flow cell.

Description of Related Art

Optical analysis of samples contained in flow cells, such aslaser-induced fluorescence, involves illuminating biological sampleswith laser light in order to test samples which may, for example, betagged with fluorescent dyes. Fluorescent dyes absorb light at certainwavelengths and in turn emit their fluorescence energy at a differentwavelength. This emission can be detected to ascertain properties of thesample contained in the flow cell. Existing systems for fluorescentanalysis of flow cells, however, suffer from various drawbacks, such asmeasurement error.

SUMMARY

Embodiments described herein have several features, no single one ofwhich is solely responsible for their desirable attributes. Withoutlimiting the scope of the inventions as expressed by the claims, some ofthe advantageous features will now be discussed briefly.

Various aspect of the disclosure can relate to an illumination system.The illumination system can include a light source that can beconfigured to provide a combined beam of light having first light of afirst wavelength and second light of a second wavelength. The firstlight and the second light can be substantially coaxial. In someembodiments, the illumination system can be configured to receive acombined beam of light that has first light of a first wavelength andsecond light of a second wavelength. The illumination system can includean optical assembly, which can include: a first surface that can beconfigured to receive the combined beam of light and to transmit thefirst light of the first wavelength and the second light of the secondwavelength; a second surface that can be configured to transmit thefirst light of the first wavelength along a first optical path and toreflect the second light of the second wavelength along a second opticalpath; a third surface that can be configured to reflect the first lightof the first wavelength (e.g., where the second optical path of thesecond light does not intersect the third surface); a fourth surfacethat can be configured to reflect the second light of the secondwavelength (e.g., where the first optical path of the first light doesnot intersect the fourth surface); a fifth surface that can beconfigured to reflect the first light of the first wavelength and totransmit the second light of the second wavelength; and a sixth surfacethat can be configured to output a first beam of light having the firstlight of the first wavelength from a first location on the sixthsurface, and to output a second beam of light having the second light ofthe second wavelength from a second location on the sixth surface thatis offset from the first location. In some implementations, the firstbeam of light and the second beam of light can have a converging anglebetween the first beam and the second beam. In some cases, theillumination system can include a cylindrical lens array positioned toreceive the first beam of light and the second beam of light. Thecylindrical lens array can be configured to alter a distribution oflight of the first beam to output a first substantially flat-topdistribution of light, and to alter a distribution of light of thesecond beam to output a second substantially flat-top distribution oflight. The illumination system can include an objective lens system thatcan be positioned to receive the light from the cylindrical lens array.The objective lens system can be configured to output a first flat-topoutput line of the first wavelength at a sample plane, and to output asecond flat-top output line of the second wavelength at the sampleplane. The second flat-top output line can be offset from the firstflat-top output line.

The illumination system can include a beam expander between the opticalassembly and the cylindrical lens array. The illumination system caninclude a cylindrical beam expander, which can be positioned to receivethe first beam of light and the second beam of light output from theoptical assembly. The cylindrical beam expander can be configured toexpand the first beam of light and the second beam of light in a firstaxis. The cylindrical beam expander can be configured to output theexpanded first beam of light and the expanded second beam of light tothe cylindrical lens array. The cylindrical lens array can be configuredto alter the distribution of light of the first and second beams oflight in a second axis that is substantially orthogonal to the firstaxis.

The light source can include a first laser configured to output thefirst light of the first wavelength, a second laser configured to outputthe second light of the second wavelength, and a beam combinerconfigured to receive the first light from the first laser and thesecond light from the second laser and to output the combined beam oflight. The illumination system can include an optical fiber that outputsthe first light of the first wavelength and the second light of thesecond wavelength. The illumination system can include a collimatingoptical element that can be positioned to receive the first light andthe second light output from the optical fiber, and can be configured tooutput the combined beam of light that is more collimated than the lightoutput by the optical fiber. The illumination system can include a firstlaser configured to output the first light of the first wavelength, asecond laser configured to output the second light of the secondwavelength, and one or more optical elements configured to couple thefirst light and the second light into the optical fiber.

The illumination system can include a mirror system positioned toreceive the first light and the second light. The mirror system can bedriven to provide a moving light beam. The mirror system can include aMEMS mirror device.

The combined beam of light provided by the light source can have thirdlight of a third wavelength. The optical assembly can include a seventhsurface configured to transmit the first light of the first wavelengthand to reflect the third light of the third wavelength along a thirdoptical path. The optical assembly can include an eighth surfaceconfigured to transmit the second light of the second wavelength and toreflect the third light of the third wavelength to output a third beamof light, such as from a third location on the sixth surface that isoffset from the first location and the second location. The cylindricallens array can be positioned to receive the third beam of light, and thecylindrical lens array can be configured to alter a distribution oflight of the third beam to output a third substantially flat-topdistribution of light. The objective lens system can be positioned toreceive the third light from the cylindrical lens array. The objectivelens system can be configured to output a third flat-top output line ofthe third wavelength at the sample plane. The third flat-top output linecan be offset from the first flat-top output line and from the secondflat-top output line.

The third location can be between the first location and the secondlocation. The third beam of light can be substantially parallel to thecombined beam of light. The first beam of light and the second beam oflight can converge toward the third beam of light. The third flat-topoutput line can be between the first flat-top output line and the secondflat-top output line.

The optical assembly includes a first polychroic filter at the secondsurface that can be configured to transmit the first light of the firstwavelength, to reflect the second light of the second wavelength, and totransmit the third light of the third wavelength. The optical assemblycan include a second polychroic filter at the fifth surface that can beconfigured to reflect the first light of the first wavelength, totransmit the second light of the second wavelength, and to transmit thethird light of the third wavelength. The optical assembly can include athird polychroic filter at the seventh surface that is configured totransmit the first light of the first wavelength and to reflect thethird light of the third wavelength. The optical assembly can include afourth polychroic filter at the eighth surface that is configured totransmit the second light of the second wavelength and to transmit thethird light of the third wavelength.

The objective lens system can include comprises an objective stoppositioned so that the first beam of light and the second beam of lightcross at the objective stop. The objective lens system can be atelecentric lens system. The objective lens system can be configured tooutput substantially parallel beams of light to produce the firstflat-top output line and the second flat-top output line.

The second surface can be angled relative to the combined beam of lightby 45 degrees. The fifth surface can be angled relative to the combinedbeam of light by 45 degrees. The third surface can be angled relative tothe combined beam of light by a first angle that is not 45 degrees. Thefourth surface can be angled relative to the combined beam of light by asecond angle that is not 45 degrees. The first angle can differ from 45degrees by about 0.1 degree to about 1 degree. The second angle candiffer from 45 degrees by about 0.1 degree to about 1 degree. An anglebetween the first beam of light and the second beam of light can bebetween about 1 mrad and about 30 mrad.

The optical assembly can include a first polychroic filter at the secondsurface that is configured to transmit the first light of the firstwavelength and to reflect the second light of the second wavelength. Theoptical assembly can include a second polychroic filter at the fifthsurface that is configured to reflect the first light of the firstwavelength and to transmit the second light of the second wavelength.

The optical assembly can be a prism assembly. The optical assembly caninclude a plurality of polychroic plates.

Various aspect of the disclosure can relate to an optical system, whichcan include a polychroic optical assembly that can be configured toreceive a combined beam of light that includes first light of a firstwavelength and second light of a second wavelength. The polychroicoptical assembly can be configured to output a first beam of the firstlight of the first wavelength and a second beam of the second light ofthe second wavelength. In some implementations, the optical system caninclude one or more optical elements that can be configured alter adistribution of light of the first beam to output a first output line ata sample plane, and to alter a distribution of light of the second beamto output a second output line at the sample plane. The second outputline can be offset from the first output line.

The polychroic optical assembly can include a prism assembly with aplurality of prism elements. The optical assembly can include aplurality of polychroic plates. The first output line can have asubstantially flat-top distribution of light along its elongate axis.The second output line can have a substantially flat-top distribution oflight along its elongate axis.

The optical system can include a light source configured to provide thecombined beam of light. The optical system can include a first laserconfigured to output the first light of the first wavelength, a secondlaser configured to output the second light of the second wavelength,and a beam combiner configured to receive the first light from the firstlaser and the second light from the second laser and to output thecombined beam of light. The optical system can include an optical fiberthat outputs the first light of the first wavelength and the secondlight of the second wavelength, and a collimating optical elementpositioned to receive the first light and the second light output fromthe optical fiber. The collimating optical element can be configured tooutput the combined beam of light that is more collimated than the lightoutput by the optical fiber. The optical system can include a firstlaser that can be configured to output the first light of the firstwavelength, a second laser configured to output the second light of thesecond wavelength, and one or more optical elements configured to couplethe first light and the second light into the optical fiber.

The optical system can include a mirror system positioned to receive thefirst light and the second light. The mirror system can be movable andin some cases can be driven to reduce speckle. The mirror system caninclude a MEMS mirror device.

The one or more optical elements can include a cylindrical lens array,which can be positioned to receive the first beam of light and thesecond beam of light. The cylindrical lens array can be configured toalter the distribution of light of the first beam, and/or to alter thedistribution of light of the second beam. The one or more opticalelements can include an objective lens system positioned to receive thelight output from the cylindrical lens array. The objective lens systemcan be configured to output a first flat-top output line of the firstwavelength at a sample plane, and to output a second flat-top outputline of the second wavelength at the sample plane. The second flat-topoutput line can be offset from the first flat-top output line. Theoptical system can include a beam expander, which can be positioned toreceive the first beam of light and the second beam of light output fromthe polychroic optical assembly. The beam expander can be a cylindricalbeam expander configured to expand the first beam of light and thesecond beam of light in a first axis. The first axis can be orthogonalto an elongate axis of the first output line and/or orthogonal to anelongate axis of the second output line.

The polychroic optical assembly can be configured to output the firstbeam of light and the second beam of light offset from each other and/orconverging. The one or more optical elements can include an objectivelens system that includes an objective stop positioned so that the firstbeam of light and the second beam of light cross at the objective stop.The one or more optical elements include a telecentric objective lenssystem. The one or more optical elements include an objective lenssystem that is configured to output substantially parallel beams oflight to produce the first output line and the second output line. Anangle between the first beam of light and the second beam of light isbetween about 1 mrad and about 30 mrad.

The polychroic optical assembly can include a first surface that can beconfigured to receive the combined beam of light and to transmit thefirst light of the first wavelength and the second light of the secondwavelength; a second surface that can be configured to transmit thefirst light of the first wavelength and to reflect the second light ofthe second wavelength; a third surface that can be configured to reflectthe first light of the first wavelength; a fourth surface that can beconfigured to reflect the second light of the second wavelength; a fifthsurface that can be configured to reflect the first light of the firstwavelength and to transmit the second light of the second wavelength;and a sixth surface that can be configured to output the first beam ofthe first light of the first wavelength and to output a second beam ofthe second light having the second wavelength. In some embodiments, thefirst surface and/or the sixth surface can be omitted. The polychroicoptical assembly can be configured to output the first beam of lightfrom a first location (e.g., on the sixth surface), and to output thesecond beam of light from a second location (e.g., on the sixth surface)that is offset from the first location. The polychroic optical assemblycan be configured to output the first beam of light and the second beamof light so that the first beam of light and the second beam of lightare converging.

The combined beam of light can have a third light of a third wavelength.The polychroic optical assembly can include a seventh surface that canbe configured to transmit the first light of the first wavelength and toreflect the third light of the third wavelength. The polychroic opticalassembly can include an eighth surface that can be configured totransmit the second light of the second wavelength and to reflect thethird light of the third wavelength, such as to output a third beam oflight from the polychroic optical assembly. The one or more opticalelements can be configured to alter a distribution of light of the thirdbeam to output a third output line at the sample plane. The third outputline can be offset from the second output line and the first outputline. The third output line can be between the first output line and thesecond output line. The third beam of light can be substantiallyparallel to the combined beam of light. The first beam of light and thesecond beam of light can converge toward the third beam of light.

The second surface can be angled relative to the combined beam of lightby 45 degrees. The fifth surface can be angled relative to the combinedbeam of light by 45 degrees. The third surface can be angled relative tothe combined beam of light by a first angle that is not 45 degrees. Thefourth surface can be angled relative to the combined beam of light by asecond angle that is not 45 degrees. The first angle can differ from 45degrees by about 0.1 degree to about 1 degree. The second angle candiffer from 45 degrees by about 0.1 degree to about 1 degree. Thepolychroic optical assembly can include a first polychroic filter at thesecond surface that is configured to transmit the first light of thefirst wavelength and to reflect the second light of the secondwavelength. The polychroic optical assembly can include a secondpolychroic filter at the fifth surface that is configured to reflect thefirst light of the first wavelength and to transmit the second light ofthe second wavelength.

In some embodiments, the polychroic optical assembly can include a firstpolychroic filter at the second surface that is configured to transmitthe first light of the first wavelength, to reflect the second light ofthe second wavelength, and to transmit the third light of the thirdwavelength. The polychroic optical assembly can include a secondpolychroic filter at the fifth surface that is configured to reflect thefirst light of the first wavelength, to transmit the second light of thesecond wavelength, and to transmit the third light of the thirdwavelength. The polychroic optical assembly can include a thirdpolychroic filter at the seventh surface that is configured to transmitthe first light of the first wavelength and to reflect the third lightof the third wavelength. The polychroic optical assembly can include afourth polychroic filter at the eighth surface that is configured totransmit the second light of the second wavelength and to transmit thethird light of the third wavelength.

The polychroic optical assembly can include a first polychroic surfaceconfigured to transmit the first light of the first wavelength and toreflect the second light of the second wavelength. The polychroicoptical assembly can include a second polychroic surface configured toreflect the first light of the first wavelength and to transmit thesecond light of the second wavelength. The polychroic optical assemblycan include a surface configured to receive the first light of the firstwavelength from the first polychroic surface, and to redirect the firstlight toward the second polychroic surface. The polychroic opticalassembly can include a surface configured to receive the second light ofthe second wavelength from the first polychroic surface, and to redirectthe second light toward the second polychroic surface. The polychroicoptical assembly can be configured to output the first beam of lightfrom a first location, and to output the second beam of light from asecond location that is offset from the first location. The polychroicoptical assembly can be configured to output the first beam of light andthe second beam of light so that the first beam of light and the secondbeam of light are angled to converge towards each other. In someembodiments, the combined beam of light can have third light of a thirdwavelength. The polychroic optical assembly can include a thirdpolychroic surface configured to transmit the first light of the firstwavelength and to reflect the third light of the third wavelength, and afourth polychroic surface configured to transmit the second light of thesecond wavelength and to reflect the third light of the thirdwavelength, such as to output a third beam of light from the polychroicoptical assembly. The one or more optical elements can be configured toalter a distribution of light of the third beam to output a third outputline (e.g., at the sample plane). The third output line can be offsetfrom the second output line and the first output line. The third outputline can be between the first output line and the second output line.The third beam of light can be substantially parallel to the combinedbeam of light. The first beam of light and the second beam of light canbe angled to converge toward the third beam of light. The polychroicoptical assembly can include a first polychroic filter that isconfigured to transmit the first light of the first wavelength, toreflect the second light of the second wavelength, and to transmit thethird light of the third wavelength. The polychroic optical assembly caninclude a second polychroic filter that can be configured to reflect thefirst light of the first wavelength, to transmit the second light of thesecond wavelength, and to transmit the third light of the thirdwavelength. The polychroic optical assembly can includes a thirdpolychroic filter that is configured to transmit the first light of thefirst wavelength and to reflect the third light of the third wavelength.The polychroic optical assembly can includes a fourth polychroic filterthat can be configured to transmit the second light of the secondwavelength and to transmit the third light of the third wavelength.

Various aspects of the disclosure can relate to a prism assembly, whichcan include a first prism that has a first surface configured to receivea combined beam of light into the first prism. The combined beam oflight can include a first wavelength of light and a second wavelength oflight. The first prism can include a second surface configured toreceive the combined beam of light, and a third surface. A second prismcan include a first surface, and a first interface can couple the secondsurface of the first prism to the first surface of the second prism. Thefirst interface can be configured to transmit the first wavelength oflight into the second prism and to reflect the second wavelength oflight toward the third surface of the first prism. The second prism canhave a second surface that can be configured to reflect the firstwavelength of light. The second prism can have a third surfaceconfigured to receive the first wavelength of light reflected by thesecond surface of the second prism. A third prism can include a firstsurface. A second interface can couple the third surface of the firstprism to the first surface of the third prism. The second interface canbe configured to transmit the second wavelength of light into the thirdprism. The third prism can include a second surface that can beconfigured to reflect the second wavelength of light. The third prismcan have a third surface configured to receive the second wavelength oflight reflected by the second surface of the third prism. A fourth prismcan include a first surface. A third interface can couple the thirdsurface of the second prism to the first surface of the fourth prism.The third interface can be configured to transmit the first wavelengthof light into the fourth prism. The fourth prism can have a secondsurface, and a fourth interface can couple the third surface of thethird prism to the second surface of the fourth prism. The fourthinterface can be configured to reflect the first wavelength of light andto transmit the second wavelength of light into the fourth prism. Thefourth prism can have a third surface that can be configured to output afirst beam of the first wavelength of light and to output a second beamof the second wavelength of light.

The second prism can include a fourth side extending between the firstside and the second side. The third prism can include a fourth sideextending between the second side and the third side. The third prismcan include includes two prism elements.

The second surface of the third prism can be angled relative to thefirst interface by about 0.1 degree to about 1 degree, although variousother angles can be used, as discussed herein. The second surface of thesecond prism can be angled relative to the fourth interface by about 0.1degree to about 1 degree, although various other angles can be used, asdiscussed herein. The prism assembly can include a first polychroicfilter positioned at the first interface and configured to transmit thefirst wavelength of light and to reflect the second wavelength of light.The prism assembly can include a second polychroic filter positioned atthe fourth interface and configured to reflect the first wavelength oflight and to transmit the second wavelength of light.

Various aspects of the disclosure can relate to an illumination system.The illumination system can include a light source that can beconfigured to provide a combined beam of light having first light of afirst wavelength and second light of a second wavelength. The firstlight and the second light can be substantially coaxial. In someembodiments, the illumination system can be configured to receive acombined beam of light that has first light of a first wavelength andsecond light of a second wavelength. The illumination system can includean optical assembly, which can include a first surface that can beconfigured to transmit the first light of the first wavelength along afirst optical path and to reflect the second light of the secondwavelength along a second optical path; a second surface that can beconfigured to reflect the first light of the first wavelength (e.g.,where the second optical path of the second light does not intersect thesecond surface); a third surface configured to reflect the second lightof the second wavelength (e.g., where the first optical path of thefirst light does not intersect the third surface); and a fourth surfaceconfigured to reflect the first light of the first wavelength and totransmit the second light of the second wavelength. The optical assemblycan be configured to output a first beam of light having the first lightof the first wavelength from a first location, and to output a secondbeam of light having the second light of the second wavelength from asecond location that can be offset from the first location. The firstbeam of light and the second beam of light can have a converging anglebetween the first beam and the second beam. The illumination system caninclude a cylindrical lens array positioned to receive the first beam oflight and the second beam of light. The cylindrical lens array can beconfigured to alter a distribution of light of the first beam to outputa first substantially flat-top distribution of light. The cylindricallens array can be configured to alter a distribution of light of thesecond beam to output a second substantially flat-top distribution oflight. The illumination system can include an objective lens system,which can be positioned to receive the light from the cylindrical lensarray, and can be configured to output a first flat-top output line ofthe first wavelength at a sample plane, and to output a second flat-topoutput line of the second wavelength at the sample plane. The secondflat-top output line can be offset from the first flat-top output line.

Various aspects of the disclosure can relate to an optical assembly,which can include a first surface that can be configured to receive acombined beam of light having first light of a first wavelength andsecond light of a second wavelength, transmit the first light of thefirst wavelength along a first optical path, and reflect the secondlight of the second wavelength along a second optical path. The opticalassembly can include a second surface that can be configured to reflectthe first light of the first wavelength. The optical assembly caninclude a third surface that can be configured to reflect the secondlight of the second wavelength. The optical assembly can include afourth surface that can be configured to receive the first light of thefirst wavelength that was reflected by the second surface, reflect thefirst light of the first wavelength to produce a first beam of lighthaving the first light of the first wavelength, receive the second lightof the second wavelength that was reflected by the third surface, andtransmit the second light of the second wavelength to produce a secondbeam of light having the second light of the second wavelength.

The optical assembly can be configured to output the first beam of lighthaving the first light of the first wavelength from a first location,and to output the second beam of light having the second light of thesecond wavelength from a second location that is offset from the firstlocation. The first beam of light and the second beam of light can havea converging angle between the first beam and the second beam. In someembodiments, the second optical path of the second light does notintersect the second surface, and/or the first optical path of the firstlight does not intersect the third surface.

The optical assembly can include a light source configured to providethe combined beam of light having first light of a first wavelength andsecond light of a second wavelength. The first light and the secondlight can be substantially coaxial. The light source or optical assemblycan include an optical fiber that can be configured to output the firstlight of the first wavelength and the second light of the secondwavelength, and a collimating optical element that can be positioned toreceive the first light and the second light output from the opticalfiber, and can be configured to output the combined beam of light thatis more collimated than the light output by the optical fiber. The lightsource can include one or more lasers to produce the first light of thefirst wavelength and the second light of the second wavelength. Thelight source can include one or more optical elements configured tocouple the first light and the second light into the optical fiber. Theoptical assembly or other systems disclosed herein can include anoptical fiber to provide light and a driver configured to vibrate theoptical fiber, such as to reduce speckle.

The optical assembly can include a cylindrical lens array positioned toreceive the first beam of light and the second beam of light. Thecylindrical lens array can be configured to alter a distribution oflight of the first beam to output a first substantially flat-topdistribution of light. The cylindrical lens array can be configured toalter a distribution of light of the second beam to output a secondsubstantially flat-top distribution of light. The optical assembly caninclude an objective lens system, which can be positioned to receive thelight from the cylindrical lens array, to output a first flat-top outputline of the first wavelength at a sample plane, and to output a secondflat-top output line of the second wavelength at the sample plane. Thesecond flat-top output line can be offset from the first flat-top outputline. The objective lens system can include an objective stop positionedso that the first beam of light and the second beam of light cross atthe objective stop. The optical assembly can include a beam expanderpositioned to receive the first beam of light and the second beam oflight, and can be configured to expand the first beam of light and thesecond beam of light in a first axis.

The third surface can be angled relative to the first surface by about0.1 degree to about 1 degree, although various other angles can be used,as discussed herein. The second surface can be angled relative to thefourth surface by about 0.1 degree to about 1 degree, although variousother angles can be used, as discussed herein. The optical assembly caninclude a first polychroic filter that can be positioned at the firstinterface and configured to transmit the first wavelength of light andto reflect the second wavelength of light. The optical assembly caninclude a second polychroic filter that can be positioned at the fourthinterface and configured to reflect the first wavelength of light and totransmit the second wavelength of light. The optical assembly caninclude a prism assembly. The optical assembly can include a pluralityof polychroic plates. The systems disclosed herein can include anoptical fiber to provide light and a driver configured to vibrate theoptical fiber to reduce speckle.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings and the associated descriptions are provided toillustrate embodiments of the present disclosure and do not limit thescope of the claims.

FIG. 1 depicts an example embodiment of a multi-laser system.

FIG. 2 depicts another example embodiment of a multi-laser system.

FIG. 2A depicts another example embodiment of a multi-laser system inwhich the beam positioning/combining system comprises mirrors.

FIG. 2B depicts an example embodiment of a triangular prism.

FIG. 2C depicts an example embodiment of a rectangular prism.

FIG. 2D depicts an example embodiment of a multi-laser system includinga prism beam positioning/combining system.

FIG. 2E depicts an example embodiment of a prism beampositioning/combining system.

FIG. 3 depicts the front view of the system of FIG. 2 .

FIG. 4 depicts the side view of the system of FIG. 2 .

FIG. 5 depicts an example embodiment of a multi-laser system furtherincluding a plurality of beam adjusters.

FIG. 6 depicts an example embodiment of a multi-laser system furtherincluding focusing optics.

FIG. 7 depicts an example embodiment of a multi-laser system in whichthe beam adjusters comprise Risley prism pairs.

FIGS. 8A-8B depict example embodiments of a multi-laser system in whichthe beam adjusters comprise Risley prisms and plane parallel plates.

FIG. 9 depicts an example embodiment of a multi-laser system in whichthe target object comprises an optical fiber or waveguide.

FIG. 10 depicts an example embodiment of a multi-laser system in whichthe target object comprises an adjuster mount.

FIGS. 11(a)-11(n) depict example spatial arrangements of laser beams inmulti-laser systems.

FIG. 11(a) depicts parallel collimated beams with identical beamseparations, d₀.

FIG. 11(b) depicts parallel collimated beams with different beamseparations, d₁ and d₂.

FIG. 11(c) depicts converging collimated beams with identical angularseparations, θ₀.

FIG. 11(d) depicts converging collimated beams with different angularseparations, θ₁ and θ₂.

FIG. 11(e) depicts diverging collimated beams with identical angularseparations, θ₀.

FIG. 11(f) depicts diverging collimated beams with different angularseparations, θ₁ and θ₂.

FIG. 11(g) depicts co-linear collimated beams.

FIG. 11(h) depicts parallel focused beams with identical beamseparations, d₀.

FIG. 11(i) depicts parallel focused beams with different beamseparations, d₁ and d₂.

FIG. 11(j) depicts converging focused beams with identical angularseparations, θ₀.

FIG. 11(k) depicts converging focused beams with different angularseparations, θ₁ and θ₂.

FIG. 11(l) depicts diverging focused beams with identical angularseparations, θ₀.

FIG. 11(m) depicts diverging focused beams with different angularseparations, θ₁ and θ₂.

FIG. 11(n) depicts co-linear focused beams.

FIG. 12 schematically shows an optical system that can be used to directlight to samples for performing optical measurements such aslaser-induced fluorescence and spectroscopic analysis.

FIG. 13 is a cross-sectional view of an embodiment of an optical fiberarray.

FIG. 14 schematically shows another optical system that can be used todirect light to samples for performing optical measurements such aslaser-induced fluorescence and spectroscopic analysis.

FIG. 15 schematically shows another optical system that can be used todirect light to samples for performing optical measurements such aslaser-induced fluorescence and spectroscopic analysis.

FIG. 16 illustrates an embodiment of an optical system configured toanalyze a sample, for example using laser induced fluorescence.

FIG. 17 illustrates another embodiment of an optical system configuredto analyze biological and/or chemical samples using laser-inducedfluorescence.

FIG. 18 illustrates an example of a flow cell comprising an array oftiles.

FIG. 19A depicts the intensity profile for light output by an examplelight source.

FIG. 19B depicts an example of a uniform intensity profile obtained byshaping the intensity profile for light output by an example lightsource shown in FIG. 19A using a beam shaping optical system.

FIG. 20 depicts the incident path of an embodiment of an optical systemconfigured to analyze samples (e.g., biological and/or chemicalsamples), such as using laser-induced fluorescence.

FIG. 21 shows the variation of the beam diameter and the intensity noiseat the output of a dynamic diffuser as a function of the distancebetween a focusing lens and the dynamic diffuser.

FIG. 22 illustrates an embodiment of a fiber coupled illuminatorcomprising two laser diodes.

FIGS. 23A-23E show the variation in the beam profile at the output ofthe dynamic diffuser as the spacing between the dynamic diffuser and afocusing lens is increased from a value less than the focal distance toa value greater than the focal distance. FIG. 23D shows the beam profilewith the spacing between the dynamic diffuser and focusing lens equal tothe focal distance.

FIG. 24 is a flow diagram illustrating a method of illuminating atarget.

FIG. 25 illustrates an embodiment of an optical system configured togenerate a flat top line beam for analyzing samples (e.g., biologicaland/or chemical samples) using laser-induced fluorescence, the opticalsystem including a MEMS mirror and a cylindrical lens array (CLA), wherethe MEMS mirror is positioned at a 45° angle relative to incident lightreceived at the MEMS mirror from the focusing lens.

FIG. 26 is a diagram illustrating an example of the X and Y-coordinatebeam profile at the sample plane of the optical system of FIG. 25 , theMEMS mirror being positioned at a 45° angle relative to incident lightreceived at the MEMS mirror from the focusing lens.

FIG. 27 is a graph illustrating an example of irradiance along thelong-axis beam profile (e.g., the Y coordinate value) at the sampleplane of the system of FIG. 25 when the MEMS mirror is positioned at a45° angle relative to incident light received at the MEMS mirror fromthe focusing lens.

FIG. 28 is a graph illustrating an example of irradiance along theshort-axis beam profile (e.g., the X coordinate value) at the sampleplane of the system of FIG. 25 , the MEMS mirror being positioned at a45° angle relative to incident light received at the MEMS mirror fromthe focusing lens.

FIG. 29 illustrates the incident light path of an embodiment of theoptical system of FIG. 25 , where the MEMS mirror is positioned at a45.5° angle relative to incident light received at the MEMS mirror fromthe focusing lens.

FIG. 30 is a diagram illustrating an example of the X and Y-coordinatebeam profile at the sample plane of the optical system of FIG. 29 , theMEMS mirror being positioned at a 45.5° angle relative to incident lightreceived at the MEMS mirror from the focusing lens.

FIG. 31 is a graph illustrating an example of irradiance along thelong-axis beam profile (e.g., the Y coordinate value) at the sampleplane of the system of FIG. 29 , the MEMS mirror being positioned at a45.5° angle relative to incident light received at the MEMS mirror fromthe focusing lens.

FIG. 32 is a graph illustrating an example of irradiance along theshort-axis beam profile (e.g., the X coordinate value) at the sampleplane of the system of FIG. 29 , the MEMS mirror being positioned at a45.5° angle relative to incident light received at the MEMS mirror fromthe focusing lens.

FIG. 33 illustrates the incident light path of an embodiment of theoptical system of FIG. 25 , where the MEMS mirror is positioned at a 46°angle relative to incident light received at the MEMS mirror from thefocusing lens.

FIG. 34 is a diagram illustrating an example of the X and Y-coordinatebeam profile at the sample plane of the optical system of FIG. 29 , theMEMS mirror being positioned at a 46° angle relative to incident lightreceived at the MEMS mirror from the focusing lens.

FIG. 35 is a graph illustrating an example of irradiance along thelong-axis beam profile (e.g., the Y coordinate value) at the sampleplane of the system of FIG. 29 , the MEMS mirror being positioned at a46° angle relative to incident light received at the MEMS mirror fromthe focusing lens.

FIG. 36 is a graph illustrating an example of irradiance along theshort-axis beam profile (e.g., the X coordinate value) at the sampleplane of the system of FIG. 29 , the MEMS mirror being positioned at a46° angle relative to incident light received at the MEMS mirror fromthe focusing lens.

FIG. 37 is a graph illustrating the Y-Centroid position of the beam (mm)on the cylindrical lens array (y-axis) as a function of the MEMS mirrortilt (x-axis).

FIG. 38 is a graph illustrating the cylindrical lens array (CLA) Y-angleof incidence (AoI) (y-axis) as a function of the MEMS mirror tilt(x-axis), showing that CLA AoI changes only slightly with MEMS mirrortilt, which prevents (or at least partially prevents) side lobes fromappearing.

FIG. 39 is a graph illustrating the Y-Centroid position (mm) of the beamon the objective lens (y-axis) as a function of the MEMS mirror tilt(x-axis).

FIG. 40 is a graph illustrating the objective lens Y-AoI (y-axis) as afunction of the MEMS mirror tilt (x-axis), showing that objective AoIchanges only slightly with MEMS mirror tilt, which is important formaintaining the position of the illumination field-of-view (FOV) as theMEMS mirror moves.

FIG. 41 illustrates the incident light path of an embodiment of anoptical system configured to analyze samples generally similar to theoptical system of FIG. 25 including having a MEMS mirror in the beampath between a focusing lens and a collimating lens, except that in thissystem the CLA has been replaced by a diffuser positioned at a distanced1 from the collimating lens and at a distance d2 from the objectivelens.

FIG. 42 illustrates an example of an embodiment of a cylindrical lensarray (CLA) that can be used in illumination systems described herein.

FIG. 43 illustrates an embodiment of an optical system configured togenerate a flat top line beam for analyzing samples the optical systemincluding a MEMS mirror and a cylindrical lens array (CLA). Unlike theoptical system of FIG. 25 , the example of an optical system in FIG. 43does not include a collimating lens between the MEMS mirror and the CLA,and does not include a focusing lens between the beam combiner and theMEMS mirror.

FIG. 44 illustrates an embodiment of an optical system configured togenerate a flat top line beam for analyzing samples (e.g., biologicaland/or chemical samples) using laser-induced fluorescence, the opticalsystem including a MEMS mirror and a cylindrical lens array (CLA),wherein an optical fiber is disposed to couple light from the lightsource to the MEMS mirror.

FIG. 45 is a schematic drawing that illustrates another embodiment of anoptical system configured to generate a flat top line beam for analyzingsamples (e.g., biological and/or chemical samples) using laser-inducedfluorescence, the optical system including a MEMS mirror and acylindrical lens array (CLA), wherein an optical fiber is disposed tocouple light from the MEMS mirror to the cylindrical lens array.

FIG. 46 is a drawing of a design of an optical system configured togenerate a flat top line beam for analyzing samples (e.g., biologicaland/or chemical samples) using laser-induced fluorescence, the opticalsystem including a MEMS mirror and a cylindrical lens array (CLA),wherein an optical fiber is disposed to couple light from the MEMSmirror to the cylindrical lens array. In this design, a rotationallysymmetric collimating lens is disposed in an optical path between theoutput of the optical fiber and the cylindrical array.

FIG. 47 is a close-up view of the MEMS mirror shown in FIG. 46 .

FIG. 48 is a diagram illustrating an example of the X and Y-coordinatebeam profile at the sample plane of the optical system of FIGS. 46 and47 .

FIG. 49 is a graph illustrating an example of irradiance along theshort-axis beam profile (e.g., the X coordinate value) at the sampleplane of the system of FIGS. 46 and 47 .

FIG. 50 is a graph illustrating an example of irradiance along thelong-axis beam profile (e.g., the Y coordinate value) at the sampleplane of the system of FIGS. 46 and 47 .

FIG. 51 is a drawing of a design of an optical system configured togenerate a flat top line beam for analyzing samples (e.g., biologicaland/or chemical samples) using laser-induced fluorescence, the opticalsystem including a MEMS mirror and a cylindrical lens array (CLA),wherein an optical fiber is disposed to couple light from the MEMSmirror to the cylindrical lens array. In this design, first and secondcylindrical collimating lens are used to collimate the light exiting theoptical fiber. The first and second cylindrical lenses have differentfocal lengths in orthogonal directions (e.g., in x and y directions) andare located at different distance from the optical fiber to producedifferent beam sizes in orthogonal directions. For example, the beamsize is larger in the x direction than in the y direction, e.g., at theCLA.

FIG. 52 is a diagram illustrating an example of the X and Y-coordinatebeam profile at the sample plane of the optical system of FIG. 51 .

FIG. 53 is a graph illustrating an example of irradiance along theshort-axis beam profile (e.g., the X coordinate value) at the sampleplane of the system of FIG. 51 .

FIG. 54 is a graph illustrating an example of irradiance along thelong-axis beam profile (e.g., the Y coordinate value) at the sampleplane of the system of FIG. 51 .

FIG. 55 is a side view of an example embodiment of an illuminationsystem.

FIG. 56 is a top view of the example embodiment of the illuminationsystem of FIG. 55 .

FIG. 57 is a side view of an example prism assembly.

FIG. 58 shows an example of a first light beam and a second light beamoutput from a prism element of the prism assembly.

FIG. 59 shows an example of a first line and a second line produced atthe sample plane.

FIG. 60 is a diagram illustrating an example of the X and Y-coordinatebeam profile of the first line of FIG. 59 .

FIG. 61 is a graph illustrating an example of irradiance along theshort-axis beam profile (e.g., the Y coordinate value) for the firstline of FIG. 59 .

FIG. 62 is a graph illustrating an example of irradiance along thelong-axis beam profile (e.g., the X coordinate value) for the first lineof FIG. 59 .

FIG. 63 is a diagram illustrating an example of the X and Y-coordinatebeam profile of the second line of FIG. 59 .

FIG. 64 is a graph illustrating an example of irradiance along theshort-axis beam profile (e.g., the Y coordinate value) for the secondline of FIG. 59 .

FIG. 65 is a graph illustrating an example of irradiance along thelong-axis beam profile (e.g., the X coordinate value) for the secondline of FIG. 59 .

FIG. 66 is a side view of another example embodiment of a prismassembly.

FIG. 67 is a perspective view of the example prism assembly of FIG. 66 .

FIG. 68 is a side view of another example embodiment of a prismassembly.

FIG. 69 is a side view of another example embodiment of a prismassembly.

FIG. 70 is a side view of an example embodiment of an illuminationsystem.

FIG. 71 is a top view of the example embodiment of the illuminationsystem of FIG. 70 .

FIG. 72 shows the first output line, the second output line, and thethird output line produced by the system of FIG. 70 .

FIG. 73 is a diagram illustrating an example of the X and Y-coordinatebeam profile of the first line of FIG. 72 .

FIG. 74 is a graph illustrating an example of irradiance along theshort-axis beam profile (e.g., the Y coordinate value) for the firstline of FIG. 72 .

FIG. 75 is a graph illustrating an example of irradiance along thelong-axis beam profile (e.g., the X coordinate value) for the first lineof FIG. 72 .

FIG. 76 is a diagram illustrating an example of the X and Y-coordinatebeam profile of the second line of FIG. 72 .

FIG. 77 is a graph illustrating an example of irradiance along theshort-axis beam profile (e.g., the Y coordinate value) for the secondline of FIG. 72 .

FIG. 78 is a graph illustrating an example of irradiance along thelong-axis beam profile (e.g., the X coordinate value) for the secondline of FIG. 72 .

FIG. 79 is a diagram illustrating an example of the X and Y-coordinatebeam profile of the third line of FIG. 72 .

FIG. 80 is a graph illustrating an example of irradiance along theshort-axis beam profile (e.g., the Y coordinate value) for the thirdline of FIG. 72 .

FIG. 81 is a graph illustrating an example of irradiance along thelong-axis beam profile (e.g., the X coordinate value) for the third lineof FIG. 72 .

FIG. 82 shows a side view of another example embodiment of a prismassembly.

FIG. 83 shows a side view of another example embodiment of a prismassembly.

FIG. 84 shows a perspective view of the example embodiment of a prismassembly of FIG. 83 .

FIG. 85 is a side view of an example embodiment of an illuminationsystem.

FIG. 86 is a top view of the example illumination system of FIG. 85 .

DESCRIPTION OF CERTAIN EMBODIMENTS

Although certain preferred embodiments and examples may be disclosedherein, inventive subject matter extends beyond the specificallydisclosed embodiments to other alternative embodiments and/or uses ofthe inventions, and to modifications and equivalents thereof. Thus, thescope of the inventions herein disclosed is not limited by any of theparticular embodiments described below. For example, in any method orprocess disclosed herein, the acts or operations of the method orprocess may be performed in any suitable sequence and are notnecessarily limited to any particular disclosed sequence.

For purposes of contrasting various embodiments with the prior art,certain aspects and advantages of these embodiments are described. Notnecessarily all such aspects or advantages are achieved by anyparticular embodiment. Thus, for example, various embodiments may becarried out in a manner that achieves or optimizes one advantage orgroup of advantages as taught herein without necessarily achieving otheraspects or advantages as may also be taught or suggested herein.

Laser Systems

FIG. 1 depicts an example embodiment of a multi-laser system. Themulti-laser system 100 depicted in FIG. 1 may comprise a mountingmechanism, mounting system (e.g., mounting alignment system), etc. for aflow cell, a flow cell mount, a light pipe, a waveguide, an opticalfiber, and/or a lab on a chip.

In some embodiments, the temperature across the enclosure may be stableover time and with changes in the ambient temperature. The constanttemperature over time may help with long term system performance. Forexample, if the enclosure temperature were to change with time, then thesystem performance would also potentially degrade with time. This couldeventually result in servicing the system, e.g., to realign the system.

The thermally stable enclosure 150 comprises a material with highthermal conductivity. In some embodiments, a material with thermalconductivity of at least about 5 W/(m K), (e.g., between about 5 W/(m K)and about 2000 W/(m K)) is used. In some embodiments, a material withthermal conductivity at least about 50 W/(m K) (e.g., between about 50W/(m K) and about 2000 W/(m K)) is used. In other embodiments, amaterial with thermal conductivity of about 375 W/(m K) or greater isused. In other embodiments, a material with thermal conductivity of atleast about 380 W/(m K) is used. In some embodiments, a material withthermal conductivity between about 125 W/(m K) and about 425 W/(m K)) isused. In some embodiments, a material with thermal conductivity betweenabout 375 W/(m K) and about 425 W/(m K)) is used. In some embodiments, amaterial with thermal conductivity between about 125 W/(m K) and about250 W/(m K)) is used. In some embodiments, a material with thermalconductivity between about 200 W/(m K) and about 250 W/(m K)) is used.In some embodiments, the material has a heat capacity corresponding tothe heat capacity of the materials described herein. The use of suchthermally conductive material helps ensure a relatively reducedtemperature variation within the enclosure 150, even when the ambienttemperature outside of the enclosure varies relatively widely.

As described more fully below, a temperature controller in thermalcontact with the enclosure adjusts the temperature of the enclosure inresponse to variations in ambient conditions. A highly thermallyconductive enclosure enables the temperature controller to more quicklyand effectively maintain the enclosure and system temperature withouttemperature gradients in response to such variations in ambientconditions. A variety of thermally conductive materials can be used(e.g., copper, aluminum, copper tungsten, ceramics, epoxy, etc.). Insome embodiments, a material with a thermal conductivity of at least 5W/(m K) may be used. In other embodiments, a material with a thermalconductivity of less than 5 W/(m K) may be used. The thermallyconductive material can be used to form the entire enclosure, or merelya portion thereof. In certain embodiments, the enclosure can include, orcan substantially include, highly thermally conductive material. Forexample, highly thermally conductive material can be used to form thetop, the bottom, or any number of the sides of the enclosure 150, or anycombination thereof. In some embodiments, a majority of the enclosure150 is made of the thermally conductive material. In some embodiments,only a relatively small portion of the enclosure 150 is made of thethermally conductive material. In some embodiments, a portion of theenclosure 150 is made of the substantially thermally conductivematerial. In some embodiments, multiple thermally conductive materialscan be used, with some areas of the enclosure 150 being more thermallyconductive than others.

The multi-laser system 100 includes a plurality of lasers 101A-101N,enclosed within the thermally stable enclosure 150. The plurality oflasers 101A-101N may comprise diode lasers, solid-state lasers,frequency-doubled lasers, and/or other types of lasers. The plurality oflasers 101A-101N output a plurality of respective laser beams 102A-102N.Each of the laser beams 102A-102N may have a wavelength different fromthe other laser beams.

As shown in FIG. 1 , the multi-laser system 100 further includes a beampositioning system 1000. To achieve a desired spatial arrangement of thelaser beams 102A-102N, the inherent laser beam boresight and centrationerrors present in lasers 101A-101N, as well angular and lateralpositioning errors present in the multi-laser system's opto-mechanicalcomponents can be compensated for. In some embodiments, the beampositioning/combining system 1000 may include mechanical and/oropto-mechanical provisions to perform such compensation.

Mechanical provisions to the laser mounting may be used to adjust theangular and/or lateral position of the lasers so that the boresight andcentration errors of the lasers 101A-101N as well as the angular andlateral positioning errors of the opto-mechanical components arecompensated for. The aligned laser beams may then be positioned orcombined by the beam positioning/combining system 1000 into a desiredspatial arrangement that a specific application requires.

Opto-mechanical provisions to the beam positioning/alignment system maybe used to allow for angular and lateral position adjustment of thelaser beams. This adjustment capability may help compensate for thelasers' boresight and centration errors as well as the angular andlateral positioning errors of the opto-mechanical components to achievea desired spatial arrangement of the laser beams.

In embodiments in which the system is used perform testing of biologicalsamples, flow cells are illuminated with laser beams. Fluorescent dyesabsorb light at certain wavelengths and in turn emit their fluorescenceenergy at a different wavelength. This emission can be detected toascertain properties of the fluid in the flow cell. Temperaturevariations may cause the wavelength and/or the intensity of light outputby the lasers to vary. Such variations in the laser beams directed intothe flow cell may cause fluctuations in output fluorescent signals,which may introduce inaccuracy in the optical measurements. Temperaturevariations and/or temperature gradients also may cause movement of theoptical elements (e.g., due to thermal expansion) and resultant shiftingof the laser beams. These pointing errors may cause the laser beams todeviate from the flow cell, such that the signal changes, or isaltogether lost, again introducing inaccuracy in the test results.

Temperature variations can result from ambient temperature fluctuations.Accordingly, reducing the temperature variation of and the presence oftemperature gradients within the laser beam system can improve theaccuracy and usability of the test results.

Various embodiments described herein may address one or more of theseproblems. FIG. 2 is a top view of another example embodiment of themulti-laser system 100. The multi-laser system 100 depicted in FIG. 2comprises a thermally stable enclosure 150 configured to mechanicallyand/or thermally couple to a flow cell 132. The thermally stableenclosure 150 helps to isolate the laser and optics within the enclosure150 from the ambient environment, which may have varying temperature. Insome embodiments, the enclosure 150 can achieve thermal stabilitythrough the use of a temperature controller, as discussed in relation toFIG. 3 below. In various embodiments, the enclosure 150 helps reducevariations in the temperature of the various components of themulti-laser system 100. By maintaining the temperature within theenclosure within a relatively small range, thermally induced laserwavelength and intensity fluctuations as well as pointing instabilitiesof the laser beams can be reduced or minimized and alignment of thelaser beams to a target object may be maintained over a range of ambienttemperatures (e.g., between about 10° C. and about 55° C.). Accordingly,the use of a thermally stable enclosure 150 may help achieve moreaccurate test results.

Some materials expand and contract when heated or cooled. Changes in theenclosure temperature or temperature variations across the enclosure canresult in a change in the relative positions of lasers, mirrors, lenses,and the target object (e.g., flow cell). Some lasers exhibit beampointing that is temperature dependent. This may be due in part to thefact that different materials are used in the construction of the laser(e.g., metals, glass, adhesives, etc.). The different materials may havedifferent thermal expansion coefficients, which may cause beamdeviations when the laser system's temperature changes. Some mirror andlens systems also show some temperature dependence for the same reason.

The multi-laser system 100 depicted in FIG. 2 includes a plurality oflasers 101, 102, 103 enclosed within the thermally stable enclosure 150.Although FIG. 2 includes three lasers, a different number of lasers canbe used. The multi-laser system 100 shown in FIG. 2 includes a 405 nmlaser, a 488 nm laser and a 635 nm laser, but other wavelengths can alsobe used (e.g., lasers having wavelengths of 375 nm, 440 nm, 515 nm, 561nm, 594 nm, 640 nm, etc.).

The plurality of lasers 101, 102, 103 output a plurality of respectivelaser beams 104, 105, 106. Laser beam 104 has a first wavelength, laserbeam 105 has a second wavelength, and laser beam 106 has a thirdwavelength. The first, second, and third wavelengths are different fromone another. In FIG. 2 , these wavelengths are 405 nm, 488 nm and 635nm, respectively, but other wavelengths can also be used (e.g., 375 nm,440 nm, 515 nm, 561 nm, 594 nm, 640 nm, etc.).

As shown in the example embodiment of FIG. 2 , the multi-laser system100 further includes a plurality of automatic power control (APC)modules 107, 108, 109. In some embodiments, the APC modules 107, 108,109 may each comprise a beamsplitter (not shown) and a photodetector(not shown) configured to sample light from the laser beams 104, 105,106, respectively, and to feed back the signal from the detector incommunication with a laser controller (not shown) to adjust the outputpower of lasers 101, 102, 103, respectively. Other approaches may alsobe possible.

Referring still to FIG. 2 , the beam positioning system comprises aplurality of wavelength selective mirrors 110, 111, 112, 113. In variousembodiments, some of the wavelength selective 110, 111, 112, 113 mirrorshave significantly different reflection or transmission properties atdifferent wavelengths. Accordingly, the wavelength selective mirrors110, 111, 112, 113 can separate or combine laser beams with differentwavelengths. In some embodiments, the mirrors 110, 112 may be broadband,for example because light is not transmitted through the mirrors 110,112. Through the use of suitable optical coatings, wavelength selectivemirrors exhibit high reflection over some range of wavelengths and hightransmission over another range of wavelengths. The wavelength selectivemirrors are appropriate for the wavelengths of the laser sources. Forexample, various of the wavelength selective mirrors will selectivelyreflect (or transmit) light propagating from one laser at a firstwavelength and not light propagating from another laser at a secondwavelength. The example embodiment of FIG. 2 depicts four wavelengthselective mirrors 110, 111, 112, 113. In other embodiments, a differentnumber of wavelength selective mirrors may be used (e.g., see FIG. 2A).In some embodiments, the wavelength selective mirrors may comprisedichroic mirrors. In some embodiments, the wavelength selective mirrorsmay comprise trichroic mirrors. In some embodiments, the wavelengthselective mirrors may comprise dichroic and trichroic mirrors. Dichroicmirrors can separate or combine lasers with two different wavelengths.In various embodiments dichroic mirrors may allow at least onewavelength to substantially or totally pass through and maysubstantially or totally reflect at least one wavelength. Trichroicmirrors can separate or combine lasers with three different wavelengths.Trichroic mirrors may be configured and/or optimized for reflectingthree wavelengths. In some embodiments, trichroic mirrors they may beconfigured such that the reflected radiation has three wavelength peaks.In some embodiments, trichroic mirrors may be configured to have or onebroad peak that covers multiple wavelengths. In other embodiments, thewavelength selective mirrors may include mirrors configured for aselective different number of wavelengths. Alternatively, non-wavelengthselective mirrors, or substantially non-wavelength selective mirrors,that do not selectively reflect (or transmit) light of one laser and notlight of another laser may be inserted in the path of the beam toredirect and/or alter the beam path or the beam. Various embodiments mayhave additional and/or other optical elements in the optical path.

The wavelength selective mirrors 110, 111, 112, 113 are configured withhighly reflective and anti-reflective coatings in accordance with thewavelengths of the plurality of laser beams 104, 105, 106. As shown inFIG. 2 , wavelength selective mirror 110 is configured to be highlyreflective of the wavelength of the laser beam 104 (e.g., 405 nm, allwavelengths); wavelength selective mirror 111 is configured to be highlyreflective of the wavelength of the laser beam 104 (e.g., 405 nm) andanti-reflective of the wavelength of the laser beam 105 (e.g., 488 nm);wavelength selective mirror 112 is configured to be highly reflective ofthe wavelength of the laser beam 106 (e.g., 635 nm, all wavelengths),and wavelength selective mirror 113 is configured to be highlyreflective of the wavelength of the laser beam 106 (e.g., 635 nm), andanti-reflective of the wavelengths of the laser beams 104 (e.g., 405 nm)and 105 (e.g., 488 nm). In other embodiments, the wavelength selectivemirrors can be configured to be highly reflective of some wavelengthsand anti-reflective of some other wavelengths in order to separate orcombine the wavelengths as necessary.

In some embodiments, this plurality of wavelength selective mirrors 110,111, 112, 113 may be supported by a plurality of respective flexuremounts (not shown). Flexure mounts are less likely to move with externalvibrations and thus are less likely to require adjustment. Flexuremounts reduce impact on the optics from shocks such as may be introducedby shipping of the system. Additionally, flexure mounts typicallyexhibit less hysteresis than rolling or sliding contacts. Flexure mountsare typically fabricated from materials which make them relatively lesssensitive to temperature variations. Flexure mounts may also be smallerthan conventional spring-loaded mounts. In some embodiments, the flexuremounts may comprise a nickel-iron alloy material for example. Othermaterials may also be used. In other embodiments, the plurality ofwavelength selective mirrors 110, 111, 112, 113 may be supported by aplurality of respective spring-loaded mirror mounts (not shown). Inother embodiments, the plurality of wavelength selective mirrors 110,111, 112, 113 may be supported by a plurality of respective glue-blockmounts (not shown).

In the multi-laser system 100 shown in FIG. 2 , three optical paths aredepicted. A first optical path at a wavelength of 405 nm originates atlaser 101, passes through the APC 107, where a portion of the signal ispicked off (e.g., by a beam splitter), is then highly reflected atwavelength selective mirrors 110 and 111 and transmitted throughwavelength selective mirror 113, and then arrives at the focusing optics117. A second optical path at a wavelength of 488 nm originates at laser102, passes through the APC 108, where a portion of the signal is pickedoff (e.g., by a beam splitter), is then transmitted through wavelengthselective mirrors 111 and 113, and then arrives at the focusing optics117. A third optical path at a wavelength of 635 nm originates at laser103, passes through the APC 109, where a portion of the signal is pickedoff (e.g., by a beam splitter), is then reflected at wavelengthselective mirrors 112 and 113, and then arrives at the focusing optics117. Propagating along these paths, laser beams 104, 105, 106, which mayhave originally been far from one another, are repositioned to be closertogether as beams 114, 115, 116 and, after the focusing optics, beams118, 119, 120, respectively. In some embodiments, the beams 118, 119,120 are parallel to one another. In other embodiments, the beams 118,119, 120 are not parallel to one another. Other mirrors and opticalcomponents (e.g., lenses, prisms, polarization rotators, waveplates,etc.) can be included to alter the laser beams and/or optical paths.

Still referring to FIG. 2 , the multi-laser system 100 further includesoptional beam focusing optics 117 to provide size reduction and/orshaping to the output laser beams 118, 119, 120. For example, thefocusing optics 117 may focus a laser beam down to a smaller spot,generally referred to herein as a focus point. Due to non-ideal opticalelements and configurations, the “focus point” where focusing opticsfocus the light the may not be an actual “point” but instead representand area larger than a point, for example, a small spot shaped area.Additionally, the focusing optics 117 may change the shape of the laserbeams. In some embodiments, for example, the laser beams 118, 119, 120can have a generally Gaussian profile, so that when illuminating a flowcell, the intensity of the light illuminating the center of the flowcell is significantly greater than the intensity of the lightilluminating the peripheral edges of the flow cell. Accordingly, thebeams of light 118, 119, 120 can be elongated (e.g., elliptical) beams,so that the relatively high intensity center regions of the light beamsextend across the entire width of the flow cell, while the relativelylow intensity outer regions of the light beams do not strike the flowcell. By using an elongated (e.g., elliptical) beam of light, a moreuniform distribution of light across the width of the flow cell or othertarget output can be achieved while illuminating a relatively smalllongitudinal area along the length of the flow cell and maintainingsubstantially uniform high light intensity.

In some embodiments, the beams 114, 115, 116 enter the beam focusingoptics 117 and can have circular cross-sections with a Gaussianfall-off. In some embodiments, the beam focusing optics 117 may includean anamorphic lens system which may produce non-rotationally symmetricor elongated beam such as a beam with elliptical cross-section and spotsize. In other embodiments, the beam focusing optics 117 may includecylindrical lenses. In some embodiments, the beam focusing optics 117may include spherical lenses. In some embodiment, the beam focusingoptics 117 may include Powell lenses (Gaussian to flat-toptransformers). In some embodiments, the beam focusing optics 117 mayinclude aspherical lenses. The focusing optics may be achromatic withreduced chromatic aberration thereby reducing positioning error whichmay otherwise result from different color laser beams. Accordingly,achromatic anamorphic optics, achromatic elliptical optics, achromaticspherical optics and achromatic aspherical optics, may be used. In someembodiments, lenses can be an anamorphic microlens array. In someembodiments, refractive and/or diffractive optics can be used to producethe elongated beams of light 118, 119, 120. Other types of optics arepossible.

In cases where the laser comprises a semiconductor laser, the laser beamoutput may already be elliptical-shaped, and optics to convert theelliptical beam into a circular beam can be excluded. In such cases,there would be no need to include anamorphic focusing optics to make theelliptical-shaped beam spherical (e.g., rotationally symmetric).Spherical or rotationally symmetric optics may be employed withoutanamorphic elements.

The output laser beams 118, 119 and 120 depicted in FIG. 2 may haverespective spot sizes of between about 55 μm and about 110 μm in onedirection and between about 5 μm and about 15 μm in another direction(e.g., perpendicular to the one direction). In other embodiments, thelaser beams may have respective spot sizes of between about 70 μm andabout 110 μm in one direction and between about 5 μm and about 15 μm inanother direction (e.g., perpendicular to the one direction). In otherembodiments, the laser beams may have respective spot sizes of betweenabout 50 μm and about 150 μm in one direction and between about 5 μm andabout 20 μm in another direction (e.g., perpendicular to the onedirection). In other embodiments, the laser beams may have spot sizes ofbetween about 55 μm and about 100 μm in one direction and between about5 μm and about 15 μm in another direction (e.g., perpendicular to theone direction). In other embodiments, the laser beams may have spotsizes of between about 70 μm and about 100 μm in one direction andbetween about 5 μm and about 15 μm in another direction (e.g.,perpendicular to the one direction). In other embodiments, the laserbeams may have respective spot sizes of between about 50 μm and about150 μm in one direction and between about 5 μm and about 20 μm inanother direction (e.g., perpendicular to the one direction). In someembodiments, the output laser beams 118, 119, 120 may have respectivespot sizes of about 80 μm in one direction and about 10 μm in anotherdirection (e.g., perpendicular to the one direction). In otherembodiments, the output laser beams 118, 119, 120 may have respectivespot sizes of about 100 μm in one direction and about 10 μm in anotherdirection (e.g., perpendicular to the one direction). The directions maycorrespond to major and minor axes of an ellipse for a beam with anelliptical cross-section and spot shape. Other sizes and shapes arepossible for the light beams.

Still referring to FIG. 2 , the multi-laser system 100 includes couplingto a flow cell 132. The multi-laser system 100 can include an outputwindow 121 that allows the beams of light 118, 119, 120 to exit theenclosure 150. The output window 121 can be made from, for example,fused silica, glass, acrylic, or a variety of other transparentmaterials (e.g., plastic). In some embodiments, the enclosure 150includes an aperture 122 in a wall thereof and the output window 121comprises a transparent window pane 124 positioned over the aperture122. The window pane 124 can be made from, for example, fused silica,glass, acrylic, or a variety of other transparent materials (e.g.,plastic). The aperture 122 and window pane 124 can assume a variety ofshapes, but in some embodiments they are rectangular, circular, orelliptical. The window 121 can be attached to the enclosure 150 by aplurality of fasteners such as bolts 126. In FIG. 2 , only two bolts 126are shown, but in some embodiments, additional bolts can be positionedalong the edges of the window 121. In some embodiments, the window 121can include a flange for mounting the window. The flange may have aplurality of through holes through which fasteners (e.g., bolts 126) canpass to secure the window 121 to the enclosure 150. A seal 128 (e.g., anO-ring) can be positioned between the enclosure 150 and the window 121.The bolts 126 can be tightened, causing the O-ring 128 to be compressedbetween the enclosure 150 and the window 121. In some embodiments, theO-ring 128 produces a hermetic seal. Other approaches can be used tofasten the window 121 to the enclosure 150. The window 121 can besecured to the enclosure 150 by an adhesive, epoxy, or cement.

In some embodiments, the seal described may produce a hermetic seal. Ahermetic seal may help reduce particles and contamination from outsidethe enclosure. A hermetic seal may also help to prevent or reduce theflow of air currents and thus prevent or reduce the flow of ambienttemperature changes into the enclosure. This in turn may help reducetemperature instability within the enclosure. In some of the embodimentsdiscussed above, the entire enclosure 150 is hermetically sealed fromthe ambient air. Thus, the interior of the enclosure 150 is isolatedfrom air currents which can cause temperature variation, and theinternal optical elements are protected from external contaminants. Insome embodiments a getter (not shown) is located inside the enclosure150 which can reduce contaminant particles or chemical species.Additionally, a desiccant (not shown) can be positioned inside theenclosure 150 to reduce moisture.

Although FIG. 2 shows a single output window, multiple output windowscan be used. For example, each beam of light 118, 119, 120 can exit theenclosure 150 via a respective output window. In some embodiments, it isdesirable that as much as possible of the enclosure 150 comprise thethermally conductive material, to better achieve temperature uniformity.Accordingly, the output windows can be separated by thermally conductivematerial and can cover only as much area as necessary to allow lightbeams 118, 119, 120 to leave the enclosure 150. However, in someembodiments, a single output window is easier and less expensive toconstruct.

The multi-laser system 100 can include a flow cell connector (not shown)that is mechanically and thermally coupled to the enclosure 150, and theflow cell connector is configured to secure a flow cell 132 so that itintersects and maintains the alignment of the beams of light 118, 119,120. In some embodiments, the flow cell connector can permanently attachthe flow cell 132 to the enclosure 150. However, in some embodiments,the flow cell connector can allow the flow cell 132 to be removablyattached to the enclosure 150. In some embodiments, the flow cellconnector can be compatible with multiple types and/or sizes of flowcells. For example, the flow cell connector can include a clip, afriction or pressure fit coupling, a threaded portion configured toreceive a corresponding threaded portion of the flow cell 132, or avariety of other connectors known in the art or yet to be devised. Theflow cell 132 can be a capillary flow cell, and at least part of theflow cell can comprise a transparent material (e.g., fused silica orglass) that allows the light beams 118, 119, 120 to enter the flow cell132 and interact with a sample fluid contained within the flow cell 132.The flow cell 132 can be a thin hollow tube, forming a flow path thathas a diameter of about 10 μm. Other flow cell types and/or sizes can beused, and the flow cell 132 can be oriented differently than as shown inFIG. 1 . In some embodiments, the beams of light 118, 119, 120 strikethe flow cell over areas centered about 110 μm to about 140 μm apartfrom each other, and in some embodiments, about 125 μm apart from eachother. In some embodiments, the beams of light 118, 119, 120 strike theflow cell over areas centered about 100 μm to about 150 μm apart fromeach other. In some embodiments, the beams of light 118, 119, 120 strikethe flow cell over areas centered about 100 μm to about 500 μm apartfrom each other. In some embodiments, the beams of light 118, 119, 120strike the flow cell over areas centered up to about 500 μm apart fromeach other. In some embodiments, the thermal expansion coefficient ofthe thermally stable enclosure 150 matches the thermal expansioncoefficient of the flow cell 132. Matching of thermal expansioncoefficients may help reduce overall stress on the flow cell. For someforms of optical measurements, it may be desirable for the differentlaser beams to be focused to different locations in the flow cell 132 atspecific locations (e.g., areas spaced about 125 μm apart).

FIG. 2A depicts another example embodiment of a multi-laser system inwhich the beam positioning/combining system comprises mirrors. As shownin FIG. 2A, a beam positioning combiner system that employs mirrorsmounted onto a frame may be used. In various embodiments, the frames onwhich the mirrors are mountable may be adjustable, e.g., translatable,tiltable, etc. In various embodiments, the wavelength selective mirrorshave significantly different reflection or transmission properties atdifferent wavelengths. Accordingly, the wavelength selective mirrors canseparate or combine laser beams with different wavelengths.

Through the use of suitable optical coatings, wavelength selectivemirrors will selectively reflect (or transmit) light of at least onewavelength and not light of at least one other wavelength. In otherembodiments, the wavelength selective mirrors may comprise mirrors withselectivity for a different number of wavelengths. The exampleembodiment of FIG. 2A depicts a plurality of wavelength-selectivemirrors. The mirrors can be used to separate or combine lasers withdifferent wavelengths. Alternatively, non-wavelength selective mirrorsthat do not selectively reflect (or transmit) light of one laser and notlight of another laser may be inserted in the path of the beam toredirect and/or alter the beam path or the beam. Other optical elementscan also be inserted into the optical path.

The wavelength-selective mirrors 221A, 221B . . . 221N are configuredwith highly reflective and anti-reflective coatings in accordance withthe wavelengths of the plurality of laser beams 102A, 102B . . . 102N.As shown in FIG. 2A, wavelength selective mirror 221A is configured tobe highly reflective of the wavelength of the laser beams 102B through102N and anti-reflective of the wavelength of laser beam 102A;wavelength-selective mirror 221B is configured to be highly reflectiveof the wavelength of the laser beam 102B and anti-reflective of thewavelength of the laser beam 102N; and wavelength selective mirror 221Nis configured to be highly reflective of the wavelength of the laserbeam 102N. Other configurations are possible.

In the multi-laser system 100 shown in FIG. 2A, a plurality of opticalpaths are depicted. A first optical path originates at laser 101A and istransmitted through wavelength selective mirror 221A and transmittedtoward the target object 1100. A second optical path originates at laser101B, is then reflected at wavelength selective mirrors 221B and 221A,and transmitted toward the target object 1100. An n-th optical pathoriginates at laser 101N, is then reflected at wavelength selectivemirror 221N, transmitted at wavelength selective mirror 221B, reflectedat wavelength selective mirror 221A, and transmitted toward the targetobject 1100. Propagating along these paths, laser beams 102A-102N, whichmay have originally been far from one another, are repositioned to becloser together as beams 2001A-2001N.

The mirrors may be configured to adjust the position of the plurality oflaser beams to be at a certain distance of one another, for example inaddition to the spacing adjustment that may be provided by placing thelasers at different heights within the enclosure. In some embodiments,the laser beams can be positioned to be coaxial, slightly offset butparallel to each other, or slightly offset but not parallel to eachother.

FIG. 2B depicts an embodiment of a triangular prism. The prism is atransparent optical element comprising a substantially transparentoptical material. The prism has flat, polished surfaces that reflectand/or refract light. One or more of these surfaces may be coated withan optical coating such as an interference coating that is reflectiveand/or anti-reflective. In some embodiments the coating is wavelengthselective. For example, the prism may be configured to be highlyreflective for certain wavelengths (e.g., of a first laser), and highlyanti-reflective for other wavelengths (e.g., of a second laser). Theexact angles between the surfaces depend on the application. As shown,the triangular prism generally has a triangular base and rectangularsides. Prisms may be made out of glass, or any material that istransparent to the wavelengths for which they are designed. In someembodiments, the material may include one of polymer, polycarbonate,polyethylene terephthalate, glycol-modified polyethylene terephthalate,amorphous thermoplastic, and/or other substrates. Prisms can be used toreflect light, and to split light into components with different, e.g.,wavelength, polarizations. As illustrated in FIG. 2B, a triangular prismincludes a glass surface configured to allow transmission of a laserbeam of a given wavelength. The surface may be coated with a reflectivecoating to allow for the reflection of the laser beam of a differentwavelength. In some embodiments, each of the wavelength selectivemirrors illustrated in FIGS. 2 and 2A may be replaced with a triangularprism as the one illustrated in FIG. 2B. Triangular prisms may also beused that reflect a plurality of wavelength, for example, using totalinternal reflection. Accordingly, the prisms may be used to redirectlaser beams and not for wavelength selection in various cases.

FIG. 2C depicts an embodiment of a rectangular prism. This rectangularprism comprises two triangular prisms contacted together. As illustratedin FIG. 2C, a rectangular prism may be used to deflect a beam of light,for example, by 90 degrees, although other angles are also possible. Asdescribed above, in some embodiments, prisms employ total internalreflection at the surfaces rather than for dispersion. If light insidethe prism hits one of the surfaces at a sufficiently steep angle(greater than the critical angle), total internal reflection occurs andall of the light is reflected. This makes a prism a useful substitutefor a mirror in some situations. As described above, triangular prismsor prisms having other shapes can also be used for this purpose. In someembodiments, rectangular prisms can be wavelength selective. Forexample, the interface between the two triangular prisms or prismportions that make up the rectangular prism shown in FIG. 2C can includean optical coating such as an interference coating that is wavelengthselective. In some embodiments, for example, the rectangular prismselectively reflects one laser wavelength and selectively transmitsanother wavelength. Accordingly, the rectangular prism may include oneor more coatings that are highly reflective for one or more laserwavelength. The rectangular prism may include one or more coatings thatare anti-reflective for one or more laser wavelength. In someembodiments, each of the wavelength selective mirrors illustrated inFIGS. 2 and 2A may be replaced with a rectangular prism such as the oneillustrated in FIG. 2C. Other arrangements and configurations are alsopossible. For example, a prism (e.g., a rectangular prism) may comprisetwo or more triangular or other shape prisms that are contactedtogether.

FIG. 2D depicts an embodiment of the multi-laser system including aprism or prism bar beam positioning/combining system 1000C. As shown inFIG. 2D, a prism-based beam positioning combiner system is used to allowthe lasers to be arranged in a row at one end of thetemperature-controlled enclosure. In some embodiments, the prism beampositioning/combining system may include optically contacted prismshaving one or more surfaces coated to allow for the selectivetransmission or reflection of the laser beams. By proper selection ofthe surface coatings (such as for example wavelength selectivereflective interference coatings), various lasers of differentwavelength may be combined and output from the prism beampositioning/combining system. The prism beam positioning/combiningsystem may also be configured and arranged with respect to the lasersand the respective laser beam paths such that the laser beams can bepositioned such that they are, for example, closely spaced and/orparallel or co-linear on the output side of the prism-based beampositioning combiner system. In other embodiments, the prism beampositioning/combining system can be configured to position the beams inconverging or diverging with respect to one another.

The prism illustrated in FIG. 2D may comprise a plurality of prisms orprism portions contacted or adhered together (e.g., using opticalcontact bonding, or optical adhesive at optical interfaces, and thelike) to make a monolithic multi-prism beam combiner, or an aggregatedprism. In some embodiments, a monolithic multi-prism may comprise 2, 3,4, 5, or more prism portions. For example, a monolithic multi-prism maycomprise N+1 or N+2 prism portions, where N is the number of lasers. Insome embodiments, a monolithic multi-prism may comprise 1, 2, 3, 4, ormore optical interfaces. For example, a monolithic prism may comprise Nor N+1 optical interfaces, where N is the number of lasers. In variousembodiments, one or more interface between the prism portions may bewavelength selective. For example, various of the prism portions may beconfigured to have one or more wavelength selective surfaces with one ormore highly reflective and/or an anti-reflective (e.g., interference)coatings in accordance with the wavelengths of the plurality of laserbeams 102A-102N. As shown in FIG. 2D, the wavelength selective internalsurface 3001A may be configured to be highly anti-reflective of thewavelength of the laser beam 102A and highly reflective of thewavelengths of laser beams 102B-102N. The wavelength selective internalsurface 3001B may be configured to be highly reflective of thewavelength of the laser beam 102B. The wavelength selective surfaceinternal surface 3001N may be configured to be highly reflective of thewavelength of the laser beam 102N.

In the embodiment shown in FIG. 2D, various prisms are contactedtogether (e.g., using cement, adhesive (e.g., optical adhesive), opticalcontact bonding) to form a monolithic multi-prism beam combiner or anintegrated or aggregated prism in the shape of a rectangular structureor bar having a rectangular base and rectangular sides. The differentprisms that are contacted together may have different shapes. Some ofthe prisms, for example, may have a base in the shape of a parallelogramand rectangular sides. Some of the other prisms may have differentshaped bases and rectangular sides. For example, at least one triangularprism is shown. Other shapes and configurations are also possible.

In the multi-laser system 100 shown in FIG. 2D, a plurality of opticalpaths are depicted. A first optical path originates at laser 101A, istransmitted through a prism portion to the internal surface 3001A, andthen is transmitted toward the target object 1100. A second optical pathoriginates at laser 101B, then is reflected at internal surfaces 3001Aand 3001B, and then is transmitted toward the target object 1100. Ann-th optical path originates at laser 101N, is transmitted throughinternal surfaces 101B through 101N-1, then is reflected at internalsurface 3001A, and then is transmitted toward the target object 1100.Propagating along these paths, laser beams 102A-102N, which may haveoriginally been far from one another, are repositioned to be closertogether as beams 3201A-3201N.

The prisms and interfaces therebetween within the prism-based beampositioning/combining system are configured to adjust the position ofthe plurality of laser beams to be at a certain distance from oneanother, in addition to the spacing adjustment that may be provided byplacing the lasers at different heights within the enclosure. In someembodiments, the laser beams can be positioned to be coaxial, slightlyoffset but parallel to each other, or slightly offset but not parallelto each other.

FIG. 2E depicts another embodiment of a prism beam positioning/combiningsystem. As shown in FIG. 2E, a prism-based beam positioning combinersystem is used to allow the lasers to be spread out over the surface ofthe temperature-controlled enclosure's base plate. The surfaces of theprisms may be coated to allow for the transmission or reflection of thelaser beams. The prism illustrated in FIG. 2E may comprise a pluralityof prisms or prism portions contacted or adhered together to make anaggregated prism or a monolithic multi-prism beam combiner. In variousembodiments, one or more interface between the prism portions may bewavelength selective. For example, various of the prism portions may beconfigured to have one or more wavelength selective surfaces with one ormore highly reflective and/or an anti-reflective (e.g., interference)coatings in accordance with the wavelengths of the plurality of laserbeams 102A-102N. As shown in FIG. 2E, the wavelength selective internalsurface 3601A may be configured to be highly anti-reflective of thewavelength of the laser beam 102A and highly reflective of thewavelength of laser beam 102B. The wavelength selective internal surface3601B may be configured to be highly anti-reflective of the wavelengthof the laser beams 102A and 102B and highly reflective of the wavelengthof laser beam 102C. The wavelength selective surface internal surface3601C may be configured to be highly anti-reflective of the wavelengthof the laser beams 102A, 102B, and 102C, and highly reflective of thewavelength of the laser beam 102D. The wavelength selective surfaceinternal surface 3601N may be configured to be highly anti-reflective ofthe wavelength of the laser beams 102A, 102B, 102C, through 102N-1, andhighly reflective of the wavelength of the laser beam 102N.

In the embodiment shown in FIG. 2E, various prisms are contactedtogether (e.g., using cement, adhesive (e.g., optical adhesive), opticalcontact bonding) to form an integrated or aggregated prism in the shapeof a rectangular structure or bar having a rectangular base andrectangular sides, or a monolithic multi-prism beam combiner. Thedifferent prisms that are contacted together have different shapes. Forexample, different triangular prisms are shown. Some of the prisms, forexample, may have a base in the shape of a right angle triangle andrectangular sides while other prisms may have a base in the shape of anequilateral triangle and have rectangular sides. Other shapes andconfigurations are also possible.

In the multi-laser system 100 shown in FIG. 2E, a plurality of opticalpaths are depicted. A first optical path originates at laser 101A, istransmitted through prism portions and internal surfaces 3601A, 3601B,3601C through 3601N, and is transmitted toward the target object 1100. Asecond optical path originates at laser 101B, is then reflected atinternal surface 3601A, transmitted through prism portions and internalsurfaces 3601B, 3601C through 3601N, and toward the target object 1100.A third optical path originates at laser 101C, is then reflected atinternal surface 3601B, transmitted through prism portions and internalsurfaces 3601C through 3601N, and toward the target object 1100. Afourth optical path originates at laser 101D, is reflected at internalsurface 3601C, transmitted through prism portions and internal surfaces3601D through 3601N, and toward the target object 1100. An n-th opticalpath originates at laser 101N, is reflected at internal surface 3601N,and transmitted toward the target object 1100. Propagating along thesepaths, laser beams 102A-102N, which may have originally been coming fromdifferent directions and far from one another, are repositioned to becloser together as beams 3701A-3701N. As described herein, the laserbeams 102A-102N may also be repositioned to be parallel to each other asbeams 3701A-3701N.

An embodiment of a beam combiner can include an arrangement of opticalelements, for example, one or more beam splitters and light directingelements. A beam splitter may divide the incident light into two paths,for example, one by transmission and another by reflection. Examples ofbeam splitters may be coated with a material that enables opticalelements to reflect monochromatic light and transmit light of otherwavelengths. In various embodiments of beam combiners, a beam combinermay include a wide range of aggregated prisms (or monolithic multi-prismbeam combiners). For example, a plurality of prism portions contacted orcoupled together. Aggregated prisms (or monolithic multi-prism beamcombiners) may include optical coating for example at interfaces betweenprism portions or prisms that make up the aggregated prism. Theseoptical coatings may be wavelength selective reflective coating or maybe anti-reflective (AR) coatings. One example of such an aggregatedprism comprising a plurality or prisms or prism portions contactedtogether is the X-prism. Other aggregated prisms, however, may also beused.

A multi-prism beam combiner may be more advantageous than beam combinersusing separate dichroic mirrors mounted in individual flexure mounts,mounted using a glue-block approach, or all mounted in a common mount.In a multi-prism beam combiner, all of the reflective surfaces are tiedtogether so that the number of opto-mechanical components that cancontribute to the relative movement of the laser beams with respect toeach other is greatly reduced thereby improving the system performance.Additionally, the reduced parts count and reduced complexity make forincreased ease of manufacturing and should allow for a decrease insystem size. Furthermore, the number of surfaces exposed to possiblecontamination is reduced. Also, the relatively large size of the prismcombiner compared to an individual dichroic mirror reduces the impactthat the coefficient of thermal expansion (CTE) mismatch between mostadhesives, the optics and the metal used in the optical mounts has onbeam position.

FIG. 3 depicts the front view of the multi-laser system 100 depicted inFIG. 2 . As described above, in some embodiments, the thermally stableenclosure 250 is hermetically sealed. The hermetic sealing may beprovided by o-rings 233. Again, hermetically sealing can reduceparticles and contamination from outside the enclosure. Moreover, asdescribed above, a hermetic seal may also reduce or prevent the flow ofair currents and thus prevent or reduce the flow of ambient temperaturechanges into the enclosure. This in turn may reduce temperatureinstability within the enclosure. In some embodiments, the top ofenclosure 250 may be thermally coupled, possibly with a copper braid, tothe main body of the enclosure 250 to reduce thermal effects.

As shown in FIG. 3 , the multi-laser system may further comprise atemperature controller 252. In some embodiments, the temperaturecontroller 252 may comprise a thermo electric cooler (TEC), atemperature sensor and control electronics. The TEC may pump heat fromone side to the other depending on the direction of current flow throughthe TEC. The direction of current flow may be determined by the controlelectronics. In some embodiments, for example, if the ambienttemperature were higher than the enclosure 250's set point temperaturethen the control electronics may direct current flow through the TEC sothat heat was pumped out of the enclosure 250 thereby helping maintainthe enclosure's set point temperature. In other embodiments, if theambient temperature were lower than the enclosure 250's set pointtemperature, then the control electronics may reverse the current flowthrough the TEC so that heat was pumped into the enclosure 250 againhelping maintain the enclosure's set point temperature. A temperaturecontroller 252 can be thermally coupled to the thermally stableenclosure 250. The temperature controller 252 can include a temperaturesensor (not shown) to measure the temperature of the thermally stableenclosure 250, and to provide feedback to the control electronics. Insome embodiments, the temperature sensor may comprise a thermistor. Thetemperature controller 252 may remove heat from or add heat to thethermally stable enclosure 250 in order to maintain a substantiallyconstant temperature in the thermally stable enclosure 250. The highthermal conductivity of the material of the enclosure 250 helps thetemperature controller to relatively quickly adjust the temperaturewithin the enclosure 250 in response to temperature variations outsideof the enclosure 250 and also reduce the presence of temperaturevariations across the enclosure 250.

As shown in FIG. 3 , the multi-laser system may also comprise abaseplate 260. The baseplate 260 may act as a thermal heat sink for thetemperature controller 252.

In some embodiments, the temperature within the thermally stableenclosure 250 can be held stable to within ±1° C., ±2° C., ±3° C., ±5°C., etc., for example, of a target temperature. In some embodiments, thetemperatures of the wavelength selective mirrors and the focusing opticscan be held to be within ±1° C., ±2° C., ±3° C., ±5° C., etc. of oneanother. In some embodiments, the temperature over a substantial portionof the enclosure can be held to be within ±1° C., ±2° C., ±3° C., ±5°C., etc. In some embodiments, the temperature over the entire enclosurecan be held to be within ±1° C., ±2° C., ±3° C., ±5° C., etc., forexample, of a target temperature. In some embodiments, the temperaturewithin the enclosure can be held to be within ±1° C., ±2° C., ±3° C.,±5° C., etc., for example, of a target temperature. In some embodiments,the temperature within the thermally stable enclosure 250 can be heldwithin ±1° C. of the target temperature. In some embodiments, the targettemperature can be between 10° C. and 50° C. In some embodiments, thetarget temperature can be between about 15° C. and about 45° C. In otherembodiments, the target temperature can be between about 15° C. andabout 35° C. In other embodiments, the target temperature can be betweenabout 10° C. and about 40° C. The temperature controller 252 alsomaintains the focused laser beams aligned with respect to the flow cellover a wide range of ambient temperatures. In some embodiments, therange of ambient temperatures can be between about 10° C. and about 55°C. In some embodiments, the range of ambient temperatures can be betweenabout 10° C. and about 50° C. In some embodiments, the range of ambienttemperatures can be between about 15° C. and about 45° C. In otherembodiments, the range of ambient temperatures can be between about 15°C. and about 35° C. In other embodiments, the range of ambienttemperatures can be between about 10° C. and about 40° C.

FIG. 3 also depicts that the three lasers 201, 202, and 203 may beplaced at different heights within the enclosure 250. The placement atdifferent heights may assist in positioning the focused laser beams at adesired spacing from one another at the flow cell. By disposing thelasers at different heights, the focused beams at the flow cell may beseparated by between about 110 μm and about 140 μm of one another. Insome embodiments, the focused beams may be separated by between about100 μm to about 150 μm of one another. In some embodiments, the focusedbeams may be separated by between about 100 μm to about 500 μm of oneanother. In some embodiments, the focused beams may be separated by upto about 500 μm of one another. The wavelength selective mirrors,however, can additionally be adjusted to account for the imperfection inlaser positions that may result, for example, from manufacturingtolerances. Accordingly, the wavelength selective mirrors may establishbetter positioning of the beams directed onto the flow cell.

FIG. 4 depicts the side view of the multi-laser system 100 depicted inFIG. 2 . FIG. 4 also shows the placement of the lasers at differentheights. The thermally stable enclosure 350 comprises wavelengthselective mirrors 310, 311, 312, 313 that are configured to adjust theposition of the plurality of laser beams 314, 315, 316 to be at acertain distance of one another, in addition to the spacing adjustmentthat may be provided by placing the lasers at different heights withinthe enclosure 350. In some embodiments, the laser beams can bepositioned to be coaxial, slightly offset but parallel to each other, orslightly offset but not parallel to each other. In some embodiments, theplurality of focused laser beams 318, 319, 320 may be separated by about110 μm and about 140 μm of one another. In some embodiments, theplurality of focused laser beams 318, 319, 320 may be separated by about100 μm and about 150 μm of one another. In some embodiments, theplurality of focused laser beams 318, 319, 320 may be separated by about100 μm and about 500 μm of one another. In some embodiments, theplurality of focused laser beams 318, 319, 320 may be separated by up toabout 500 μm of one another. In some embodiments, the plurality offocused laser beams 318, 319, 320 may be positioned to be at a distanceof about 125 μm of one another.

As can be seen in FIG. 4 , the thermally stable enclosure 350 comprisesa top, a bottom, and four sides. In some embodiments, the thermallystable enclosure 350 has a width of about 3 inches or less, a length ofabout 6 inches or less, and/or a height of about 2 inches or less. Inother embodiments, the length, the width, and the height of thethermally stable enclosure 350 may be relatively larger or smaller. Insome embodiments, the thermally stable enclosure 350 has a width ofabout 6 inches or less, a length of about 12 inches or less, and/or aheight of about 3 inches or less. In some embodiments, the thermallystable enclosure 350 has a volume of 36 cubic inches (in³) or less. Witha relatively small volume, the temperature controller is better able toadjust the temperature of the enclosure and system in response tovariations in ambient temperature. The temperature controller is thusable to avoid temporal variations in temperature induced by fluctuationin ambient conditions. The relatively small volume may reducetemperature instabilities within the enclosure 350 by reducingtemperature gradients across the enclosure 350. In other embodiments,the volume of the thermally stable enclosure 350 may be relativelylarger or smaller. Also shown in FIG. 4 is the flow cell connection 330,described above.

FIG. 5 depicts an example embodiment of a multi-laser system furtherincluding an optional plurality of beam adjusters 504A-504N. In variousembodiments, the boresight and centration errors of the n laser beamsand/or the angular and lateral positioning errors of the opto-mechanicalcomponents may be compensated for by using the separate beam positionadjusters 504A-504N. The adjusted laser beams may then be positionedand/or combined into a desired spatial arrangement by the beampositioning/combining system that a specific application requires.

FIG. 6 depicts the embodiment of the multi-laser system of FIG. 5further including optional focusing or beam shaping optics 117. Asdescribed in relation to FIG. 2 above, beam focusing optics or beamshaping optics may be used to provide size reduction and/or shaping tothe output laser beams. For example, the focusing/beam shaping opticsmay focus a laser beam down to a smaller spot. The focusing/beam shapingoptics may also be used to change the shape of the laser beams.

The output laser beams depicted in FIG. 6 may have respective spot sizesof between about 55 μm and about 110 μm in one direction and betweenabout 5 μm and about 15 μm in another direction (e.g., perpendicular tothe one direction). In other embodiments, the laser beams may haverespective spot sizes of between about 70 μm and about 110 μm in onedirection and between about 5 μm and about 15 μm in another direction(e.g., perpendicular to the one direction). In other embodiments, thelaser beams may have respective spot sizes of between about 50 μm andabout 150 μm in one direction and between about 5 μm and about 20 μm inanother direction (e.g., perpendicular to the one direction). In otherembodiments, the laser beams may have spot sizes of between about 55 μmand about 100 μm in one direction and between about 5 μm and about 15 μmin another direction (e.g., perpendicular to the one direction). Inother embodiments, the laser beams may have spot sizes of between about70 μm and about 100 μm in one direction and between about 5 μm and about15 μm in another direction (e.g., perpendicular to the one direction).In other embodiments, the laser beams may have respective spot sizes ofbetween about 50 μm and about 150 μm in one direction and between about5 μm and about 20 μm in another direction (e.g., perpendicular to theone direction). In some embodiments, the output laser beams 118, 119,120 may have respective spot sizes of about 80 μm in one direction andabout 10 μm in another direction (e.g., perpendicular to the onedirection). In other embodiments, the output laser beams 118, 119, 120may have respective spot sizes of about 100 μm in one direction andabout 10 μm in another direction (e.g., perpendicular to the onedirection). These may correspond to major and minor axes of an ellipsefor a beam with an elliptical cross-section and spot shape. Other sizesand shapes are possible for the light beams.

FIG. 7 depicts an example embodiment of a multi-laser system in whichthe beam adjusters 504A-504N comprise Risley prism pairs 705A-705N. Inother embodiments, other systems may be used as the separate beamposition adjusters 504A-504N. In various embodiments, the laserboresight and opto-mechanical angular errors may be compensated for byrotating the Risley prisms while the laser centration andopto-mechanical lateral positioning errors may be compensated for byadjusting the Risley prism assembly pitch, yaw, and/or separationbetween the individual prisms (e.g., by adjusting one or both of theindividual prisms). The aligned laser beams may then be positioned orcombined into a desired spatial arrangement that a specific applicationrequires by the beam positioning/combining system.

FIG. 7 depicts a Risley prism pair used with each laser beam. In otherembodiments, a different number of Risley prisms may be used. Otheroptical elements can also be inserted into the optical path.

In various embodiments, Risley prisms comprising wedged optics, usuallyused in pairs, to redirect optical beams are used. In variousembodiments, an incoming light beam enters a Risley prism pair,experiences refraction and redirection under Snell's Law, and exits theRisley prism pair. In some configuration of the Risley prisms, there isjust a translation of the output beam with respect to the input beam. Ifthe arrangement of the Risley prisms with respect to each other ischanges, the output beam may experience an elevation deviation. Theability to control azimuth may be provided by rotating the prism pairtogether. Therefore, the Risley prism pair can be used to direct a lightbeam at a variety of elevation angles and azimuthal angles.

The Risley prism pairs 705A-705N wedge angles and the azimuthal rotationbetween the prisms are determined in accordance with the respectivelaser beam 102A-102N. As shown in FIG. 6 , Risley prism pair 705A isconfigured to adjust the laser beam 102A, Risley prism pair 705B isconfigured to adjust the laser beam 102B, and Risley prism pair 705N isconfigured to adjust the laser beam 102N.

In the multi-laser system 100 shown in FIG. 7 , a plurality of opticalpaths are depicted. A first optical path originates at laser 101A,passes through the Risley prism pair 705A, where laser boresight,centration and opto-mechanical angular and lateral positioning errorsmay be compensated through adjustment of the wedge angles of and theazimuthal rotation between the prism pair 705A, and then arrives at thebeam combining/positioning system 1000. A second optical path originatesat laser 101B, passes through the Risley prism pair 705B, where laserboresight, centration and opto-mechanical angular and lateralpositioning errors may be compensated through adjustment of the wedgeangles of and the azimuthal rotation between the prism pair 705B, andthen arrives at the beam combining/positioning system 1000. An N-thoptical path originates at laser 101N, passes through the Risley prismpair 705N, where laser boresight, centration and opto-mechanical angularand lateral positioning errors may be compensated through adjustment ofthe wedge angles of and the azimuthal rotation between the prism pair705N, and then arrives at the beam combining/positioning system 1000.

Propagating along these paths, laser beams 102A-102N, which may haveoriginally been far from one another, are repositioned to be closertogether as beams 1001A-1001N. In some embodiments, the beams1001A-1001N are parallel to one another. In other embodiments, the beams1001A-1001N are not parallel to one another. Other optical components(e.g., lenses, prisms, polarization rotators, waveplates, etc.) can beincluded to alter the laser beams and/or optical paths.

FIGS. 8A-8B depict example embodiments of a multi-laser system in whichthe beam adjusters comprise Risley prisms and plane parallel plates. Inthe embodiment of FIG. 8A, a combination of Risley prism pairs 705A-705Nand glass etalon plates 707A-707N are used for the separate beamposition adjusters 504A-504N illustrated in FIG. 5 . In otherembodiments, the etalon plates may be comprised of material other thanglass. In some embodiments, the plane parallel plates may be made out ofglass, or any material that is transparent to the wavelengths for whichthey are designed. In some embodiments, the material may include one ofpolymer, polycarbonate, polyethylene terephthalate, glycol-modifiedpolyethylene terephthalate, amorphous thermoplastic, and/or othersubstrates. The etalon plates comprise plane parallel plates. Otheroptical elements may however be used in different embodiments.Adjustment of the beams may be provided for by using the combination ofRisley prism pairs as described in relation to FIG. 7 above, and glassetalon plates 707A-707N. In the embodiment of FIG. 8A, a single glassetalon plate may be used for providing correction to lateral positioningerrors in both x and y planes. In the embodiment of FIG. 8B, a separateglass etalon plate is used for correcting later positioning errors ineach (e.g., by being tiltable along one axis) of the x and y planes orin both (e.g., by being tiltable along multiple axes) of the x and yplanes.

In various embodiments, the laser boresight and opto-mechanical angularerrors may be compensated for by rotating the prisms while the lasercentration and opto-mechanical lateral positioning errors may becompensated for by adjusting the pitch and/or yaw of the paralleloptical plate. The aligned laser beams may then be positioned orcombined into a desired spatial arrangement by the beampositioning/combining system that a specific application requires.

In the multi-laser system 100 shown in FIG. 8A, a plurality of opticalpaths are depicted. A first optical path originates at laser 101A,passes through the Risley prism pair 705A, where laser boresight andopto-mechanical angular errors may be compensated through adjustment ofthe wedge angles of the prism pair 705A, passes through the glass etalonplate 707A, where laser centration and opto-mechanical lateralpositioning errors may be compensated for by adjusting the pitch and/oryaw of the glass etalon plate 707A, and then arrives at the beamcombining/positioning system 1000. A second optical path originates atlaser 101B, passes through the Risley prism pair 705B, where laserboresight and opto-mechanical angular errors may be compensated throughadjustment of the wedge angles of the prism pair 705B, passes throughthe glass etalon plate 707B, where laser centration and opto-mechanicallateral positioning errors may be compensated for by adjusting the pitchand/or yaw of the glass etalon plate 707B, and then arrives at the beamcombining/positioning system 1000. An N-th optical path originates atlaser 101N, passes through the Risley prism pair 705N, where laserboresight, and opto-mechanical angular errors may be compensated throughadjustment of the wedge angles of the prism pair 705N, passes throughthe glass etalon plate 707N, where laser centration and opto-mechanicallateral positioning errors may be compensated for by adjusting the pitchand/or yaw of the glass etalon plate 707N, and then arrives at the beamcombining/positioning system 1000.

In the multi-laser system 100 shown in FIG. 8B, a plurality of opticalpaths are depicted. A first optical path originates at laser 101A,passes through the Risley prism pair 705A, where laser boresight andopto-mechanical angular errors may be compensated through adjustment ofthe wedge angles of the prism pair 705A, passes through glass etalonplates 707A, 708A, where laser centration and opto-mechanical lateralpositioning errors may be compensated for by adjusting the pitch and/oryaw of the glass etalon plates 707A and/or 708A, and then arrives at thebeam combining/positioning system 1000. A second optical path originatesat laser 101B, passes through the Risley prism pair 705B, where laserboresight and opto-mechanical angular errors may be compensated throughadjustment of the wedge angles of the prism pair 705B, passes throughglass etalon plates 707B, 708B, where laser centration andopto-mechanical lateral positioning errors may be compensated for byadjusting the pitch and/or yaw of the glass etalon plates 707B and/or708B, and then arrives at the beam combining/positioning system 1000. AnN-th optical path originates at laser 101N, passes through the Risleyprism pair 705N, where laser boresight, and opto-mechanical angularerrors may be compensated through adjustment of the wedge angles of theprism pair 705N, passes through glass etalon plates 707N, 708N, wherelaser centration and opto-mechanical lateral positioning errors may becompensated for by adjusting the pitch and/or yaw of the glass etalonplates 707N and/or 708N, and then arrives at the beamcombining/positioning system 1000.

Propagating along these paths, laser beams 102A-102N, which may haveoriginally been far from one another, are repositioned to be closertogether as beams 1001A-1001N. In some embodiments, the beams1001A-1001N are parallel to one another. In other embodiments, the beams1001A-1001N are not parallel to one another. Other optical components(e.g., lenses, prisms, polarization rotators, waveplates, etc.) can beincluded to alter the laser beams and/or optical paths.

FIG. 9 depicts an example embodiment of a multi-laser system in whichthe target object 1100 comprises an optical fiber or waveguide. Anoptional lens 902 may be used to couple the beams into the optical fiber900. As shown in FIG. 9 , the n laser beams may be combined, forexample, by one of the embodiments for beam positioning and combiningdescribed above, or any combination of the embodiments described aboveand coupled into an optical fiber or waveguide. Both the coupling opticsand fiber or waveguide may be located inside the temperature-controlledenclosure and hard-mounted to the enclosure's temperature controlledbase plate 901.

FIG. 10 depicts an example embodiment of a multi-laser system in whichthe target object 1100 comprises an adjuster mount 1010. This adjustermount may be configured to receive an optical fiber 900. Fiber opticcoupling mounts are commercially available. In various embodiments, thefiber optic coupling mounts may comprise coupling optics mounted onefocal length away from the optical fiber input with the optical axis ofthe coupling optics co-linear with a beam that would be emitted from theoptical fiber. The coupling optics and optical fiber are mounted in ametal housing (e.g., a cylindrical housing) so that the alignmentbetween the coupling optics and optical fiber input is maintained. Thiscomponent may be a coupler/collimator assembly. In some embodiments, themounts may comprise coupling optics and fiber in a metal housing. Thecoupler/collimator assembly is inserted into a positioning mount whichis attached to the temperature-controlled enclosure. For polarizationmaintaining fibers the coupler/collimator assembly and positioningmounts may be keyed so as to provide registration between thepolarization axis of the laser beams and the polarization axis of theoptical fiber. The positioning mount may be made of metal. The mount hasmechanical adjusters that allow the pitch, yaw, and lateral position ofthe coupler/collimator assembly to be moved relative to the input laserbeams to optimize the amount of light coupled into the optical fiber. Asshown in FIG. 10 , the n laser beams may be combined, for example, byone of the embodiments for beam positioning and combining describedabove, or any combination of the embodiments described above and coupledinto the adjustor mount or an optical element such as an optical fibercoupled to the adjuster mount. The optical fiber, coupling optics andcoupling optimization hardware may be mounted at least partially on theoutside of the temperature-controlled enclosure.

FIGS. 11A-11N depict example types and spatial arrangements of laserbeams in multi-laser systems. The specific desired type and spatialarrangement of the laser beams may be application specific. The laserbeams themselves may be collimated or focused. In some applications,however, it may be desirable to focus the laser beam in order to createsmall spot sizes at the target object and, hence, to increase powerdensity or brightness on the target object. Accordingly, laser beamsthat are collimated or focused beams may be used. A plurality ofcollimated or a plurality of focused beams may be substantiallyco-linear to one another at the target object. The plurality of beamsmay alternatively be substantially parallel to one another but spacedapart from each other, or converging towards each other, or divergingaway from one another at the target object. In some embodiments, theplurality of parallel beams may have identical beam separations (such asa substantially constant separation d₀) at the target object, ordifferent beam separations (such as separations d₁, d₂, . . . ) at thetarget object. In some embodiments, the plurality of converging ordiverging beams may have identical angular separations (such as asubstantially constant separation θ₀) at the target object, or differentangular separations (such as separations θ₁, θ₂, . . . ) at the targetobject. In some embodiments, the angular separation may be less thanabout 5°.

Some examples of spatial arrangements of laser beams are depicted inFIGS. 11A-11N. While these examples illustrate spatial arrangements oftwo or three laser beams, arrangements with more than two or three laserbeams are also contemplated, having similar characteristics of theillustrated arrangements. In a first example, FIG. 11A depicts anexample of parallel, or nearly parallel, collimated beams withidentical, or nearly identical, beam separations, d₀. As used herein,the term “identical” is generally used to indicate that two things(e.g., a distance, angle, and the like) are within 3% of the measuredcharacteristic of each other, unless the context of use of the termindicates otherwise. As used herein, the term “nearly identical” isgenerally used to indicate that two things (e.g., a distance, angle, andthe like) are the within 10% of the measured characteristic of eachother, unless the context of use of the term indicates otherwise. FIG.11B depicts an example of parallel collimated beams with different beamseparations, d₁ and d₂. FIG. 11C depicts an example of convergingcollimated beams with identical angular separations, θ₀. FIG. 11Ddepicts an example of converging collimated beams with different angularseparations, θ₁ and θ₂. FIG. 11E depicts an example of divergingcollimated beams with identical angular separations, θ₀. FIG. 11Fdepicts an example of diverging collimated beams with different angularseparations, θ₁ and θ₂. FIG. 11G depicts an example of co-linearcollimated beams. FIG. 11H depicts an example of parallel focused beamswith identical beam separations, d₀. FIG. 11I depicts an example ofparallel focused beams with different beam separations, d₁ and d₂. FIG.11J depicts an example of converging focused beams with identicalangular separations, θ₀. FIG. 11K depicts an example of convergingfocused beams with different angular separations, θ₁ and θ₂. FIG. 11Ldepicts an example of diverging focused beams with identical angularseparations, θ₀. FIG. 11M depicts an example of diverging focused beamswith different angular separations, θ₁ and θ₂. FIG. 11N depicts anexample of co-linear focused beams.

FIG. 12 schematically shows an optical system 5100 that can be used todirect light to a sample for performing optical measurements such aslaser-induced fluorescence and spectroscopic analysis. The opticalsystem 5100 can include a housing 5102 enclosing an interior chamber5104. The housing 5102 can be made of a thermally conductive material.The thermally conductive material can have a thermal conductivitybetween about 50 W/(m-K) and about 2000 W/(m-K). For example, thethermally conductive material may be copper which has a thermalconductivity of about 380 W/(m-K). A variety of thermally conductivemetals can be used (e.g., copper or aluminum), as well as thermallyconductive non-metals (e.g., ceramics or epoxy). The thermallyconductive material can be used to form the entire housing, or merely aportion thereof. For example, substantially thermally conductivematerial can be used to form the top, the bottom, or any number of thesides of the housing 5102, or any combination thereof. In someembodiments, a majority of the housing 5102 is made of the substantiallythermally conductive material. In some embodiments, only a relativelysmall portion of the housing 5102 is made of the substantially thermallyconductive material. In some embodiments, a substantial portion of thehousing 5102 is made of the substantially thermally conductive material.In some embodiments, multiple substantially thermally conductivematerials can be used, with some areas of the housing 5102 being morethermally conductive than others.

In some of the embodiments discussed above, the housing is hermeticallysealed from the ambient air. Thus, the interior chamber 5104 is isolatedfrom air currents which can cause temperature variation, and theinternal optical elements are protected from external contaminants. Insome embodiments a getter (not shown) is located inside interior chamber5104 which can reduce contaminant particles or chemical species.Additionally, a desiccant (not shown) can be positioned inside theinterior chamber 5104 to reduce moisture.

A thermoelectric controller 5106 can be thermally coupled to the housing5102. The thermoelectric controller 5106 can include one or moretemperature sensors (not shown) (e.g., thermistors) to measure thetemperature of the housing 5102 and/or the temperature of the interiorchamber 5104, and a heat transfer system (not shown) for removing heatfrom or adding heat to the housing 5102 in order to maintain asubstantially constant temperature in the housing or in the interiorchamber. In some embodiments, the thermoelectric controller 5106 caninclude a cooler for removing heat (e.g., heat resulting from operationof the optical system). In some embodiments, the thermoelectriccontroller 106 can include a heater for heating the housing 5102 andinternal chamber 5104. In some embodiments, the heater can be used tomaintain the internal chamber 5104 at a temperature above theanticipated highest ambient temperature. In some embodiments, thethermoelectric controller 5106 can include a thermoelectric cooler(TEC). The heat transfer system can be coupled directly to the housing5102 and to the cooler and/or heater (e.g. TEC). In some embodiments,the temperature can be held within held within ±1° C., ±2° C., ±3° C.,±5° C., etc. of the target temperature. In some embodiments, thetemperature of the interior chamber 5104 is between 15° C. and 45° C.

In some embodiments, the housing is compact. For example, the housingmay be a size of less than 10 cubic inches. The relatively small size ofthe volume allows for rapid adjustment of temperature in response tovariations in the ambient temperature and thus more precise control ofthe temperature in the internal chamber 5104.

The optical system 5100 can include a number of optical input ports5108A-5108D. Although the embodiment shown in FIG. 12 includes fouroptical input ports, a different number of optical input ports can beused. In some embodiments, the optical input ports 5108A-5108D can besecured and hermetically sealed into respective apertures formed in thehousing 5102, and can engage optical fibers 5110A-5110D. A variety offiber connectors can be used, such as screw-type optical fiberconnectors (e.g., an FC connector), snap-type fiber connectors, or otherfiber connectors known in the art or yet to be devised. In someembodiments, the optical input ports 5108A-5108D include anangle-polished fiber connector (e.g., an FC/APC connector). In someembodiments, at least a portion of the optical input ports 5108A-5108D,such as the threading of a screw-type connector, can be integrallyformed as part of the housing 5102. The optical fibers 5110A-5110Dinclude fiber connectors (not shown) configured to securely andprecisely mate with the optical input ports 5108A-5108D so that lightcan be efficiently transferred from the optical fibers 5110A-5110D to aplurality of optical fibers 5114A-5114D within the internal chamber5104. In some embodiments, the optical fibers 5110A-5110D are singlemode optical fibers. Highly polarized light can be injected into theoptical fibers 5110A-5110D (e.g., from a diode laser), and in someapplications it can be advantageous to preserve the polarization of thelight. Accordingly, polarization-maintaining optical fibers can be used.In some embodiments different types of optical fibers can be connectedto different optical input ports 5108A-5108D. Likewise, in someembodiments, the different optical input ports 5108A-5108D can comprisedifferent types of optical connectors.

The optical fibers 5110A-5110D can be coupled to laser light sources5112A-5112D. Although the embodiment shown in FIG. 12 includes fourlasers, a different number of lasers can be used. The lasers 5112A-5112Dcan include a variety of different laser types and can provide light ofvariety of different wavelengths. The optical system 5100 shown in FIG.12 includes a 405 nm laser, a 488 nm laser, a 561 nm laser, and a 640 nmlaser, but other common wavelengths of laser light can be used (e.g.,light having a wavelength of 440 nm, 635 nm, or 375 nm). The lasers5112A-5112D can be diode lasers, diode-pumped solid state lasers,frequency doubled lasers, or other laser types that produce light usefulfor example in laser-induced fluorescence and spectroscopic analysis.Although FIG. 12 shows the lasers 5112A-5112D connected to the opticalinput ports 5108A-5108D via the optical fibers 5110A-5110D, in someembodiments the optical fibers 5110A-5110D and the lasers 5112A-5112Dcan be disconnected from the optical input ports 5108A-5108D by the userso that other lasers can be interchangeably connected to the opticalsystem 5100. Thus, the optical system 5100 is a versatile tool which auser can easily modify to utilize a wide variety of lasers withoutdifficult and time-consuming adjustments.

The optical system 5100 can include a plurality of optical fibers5114A-5114D contained within the internal chamber 5104. The opticalfibers 5114A-5114D can be optically coupled to the optical input ports5108A-5108D so that they receive light from the optical input ports5108A-5108D and direct the light into the internal chamber 5104. In someembodiments, the cores of the optical fibers 5114A-5114D can be exposedby optical input ports 5108A-5108D so that the cores of the opticalfibers 5110A-5110D can contact the cores of the optical fibers5114A-5114D directly or come in substantial proximity to the cores ofoptical fibers 5114A-5114D. As with the optical fibers 5110A-5110Ddiscussed above, the optical fibers 5114A-5114D can be single modeoptical fibers and can be polarization-maintaining optical fibers.

In some embodiments, the optical system can include a fiber supportstructure 5116 that is configured to change the pitch of the opticalfibers 5114A-5114D, bringing the output ends closer together than theinput ends. For example, the optical input ports 5108A-5108D can bespaced about 10 to 20 millimeters or more apart from each other, so thatthe user can conveniently connect and disconnect optical fibers. Theinput ends of the optical fibers 5114A-5114D, which are coupled to theoptical input ports 5108A-5108D, can be similarly distributed forexample about 10 to 20 millimeters or more apart. The fiber supportstructure 5116 can have grooves (e.g., V-grooves) defining generallyconverging pathways, and the optical fibers 5114A-5114D can be securedin the grooves by a top-plate positioned over the grooves or by anadhesive. In some embodiments, the V-grooves can be configured toprecisely hold the fibers. In some embodiments, silicon V-groovesmanufactured using silicon processing techniques (e.g., etching,photoresists, etc.) can be used to secure the optical fibers5114A-5114D. Grooves, holes, or slots for supporting the optical fibers5114A-5114D may be formed in a support material (e.g., aluminum) by amachining process, such as electrical discharge machining (EDM). Thefiber support structure 5116 can be configured to bring the opticalfibers 5114A-5114D closer together so that when the light is output fromthe optical fibers 5114A-5114D the light is emitted from nearbylocations (e.g., about 110 to 140 microns apart, and more specifically,about 125 microns apart, although other distances are possible).

FIG. 13 is a cross-sectional view (shown from the position indicated byline 2-2 in FIG. 12 ) of an embodiment of optical fibers 5214A-5214D. Asshown in FIG. 13 , the optical fibers 5214A-5214D can be single modeoptical fibers that have output ends measuring about 125 microns intotal diameter, with the core measuring about 3-4 microns in diameter.Other sizes can be used. In the embodiment shown in FIG. 13 , the outputends of the optical fibers 5214A-5214D are brought close together sothat the cladding of one optical fiber is adjacent to the cladding ofthe next optical fiber, and light is emitted by the cores of opticalfibers 5214A-5214D at locations which have centers positioned about 125microns apart. Other arrangements are possible. It should be noted thatthe drawings herein are not drawn to scale (unless otherwise indicated),and in some embodiments the tapering of the optical fibers provided bythe fiber support structure 5116 can be much more pronounced than isindicated in FIG. 12 .

In some embodiments, the fiber support structure 5116 does not bring theoptical fibers 5114A-5114D significantly closer together, but merelyorients the optical fibers 5114A-5114D so that light is emitted in adirection that causes the light to contact the optical elements5118A-5118D at a suitable angle. Other variations are possible.

Although the embodiment illustrated by FIG. 12 includes optical fibers5114A-5114D, other types of waveguides can be used (e.g., planarwaveguides). In some embodiments, the waveguides can be rigidwaveguides. The waveguides can include curved and/or linear paths. Thewaveguides can include a taper to otherwise have an output end withoutputs closer together than inputs at an input end, similar to theembodiment shown in FIG. 12 . In some embodiments, an integratedwaveguide chip is used.

Although the embodiment illustrated in FIG. 12 shows the optical fibers5110A-5110D and the optical fibers 5114A-5114D as being different setsof optical fibers, in some embodiments, the optical system can include asingle set of optical fibers that extend through the housing and coupleto the laser light sources. In these embodiments, the optical inputports 5108A-5108D can be apertures in the housing 5102 through which theoptical fibers can pass. In some embodiments, the apertures can includeseals formed around the optical fibers to hermetically seal the interiorchamber. Epoxy may be used to provide such a hermetic seal, althoughother approaches can be used. The optical fibers can include opticalconnectors (e.g., FC/APC connectors) configured to removably couple withthe laser light sources 5112A-5112D.

The optical fibers 5114A-5114D (or waveguides) emit light toward aplurality of optical elements 5118A-5118D, which convert the light intobeams of light 5120A-5120D having a suitable shape and/or size. Theoptical elements 5118A-5118D can be lenses, and can be separateindividual lenses, or they can be conjoined forming a lens array. Insome embodiments, optical elements 5118A-5118D can be compactmicrolenses. In some embodiments, a single lens can be used to produceeach of the light beams 5120A-5120D. In some applications, it can beadvantageous to produce elongated beams of light, such as beams of lighthaving a generally elliptical cross-sectional shape (shown schematicallyin FIG. 12 ). For example, the beams of light 5120A-5120D can have agenerally Gaussian profile, so that when illuminating a flow cell, theintensity of the light illuminating the center of the flow cell issignificantly greater than the intensity of the light illuminating theperipheral edges of the flow cell. Accordingly, the beams of light5120A-5120D can be elongated (e.g., elliptical) beams, so that therelatively high intensity center regions of the light beams extendacross the entire width of the flow cell, while the relatively lowintensity outer regions of the light beams do not strike the flow cell.By using an elongated (e.g., elliptical) beam of light, a more uniformlateral distribution of light across the narrow width of the flow cellcan be achieved while illuminating a relatively small longitudinal areaalong the length of the flow cell and maintaining high light intensity.In some embodiments, the elliptical light beams can have a substantiallyelliptical cross sectional shape that measure about 5 to 15 microns inone direction and 55 to 100 microns in the other direction, or morespecifically about 10 microns in one direction and about 70 microns inthe other direction. Light beams of other shapes and sizes can be used.To produce elongated (e.g., elliptical) beams of light 5120A-5120D,optical elements 5118A-5118D can be anamorphic lenses (e.g., cylindricallenses) or Powell lenses (Gaussian to flat-top transformers). In oneembodiment, optical elements 5118A-5118D can be an anamorphic microlensarray. In some embodiments, the optical elements 5118A-5118D can beachromatic lenses. In some embodiments, optical elements 5118A-5118D canbe refractive and/or diffractive optical elements used to produce theelongated beams of light 5120A-5120D. In some embodiments, the opticalelements 5118A-5119D can be located adjacent to the output ends of theoptical fibers 5114A-5114D.

The optical system 5100 can include an output window 5121 that allowsthe beams of light 5120A-5120D to exit the internal chamber 5104. Insome embodiments, the housing 5102 includes an aperture 5122 in a wallthereof and the output window 5121 comprises a transparent window pane5124, positioned over the aperture 5122. The window pane 5124 can bemade from glass or acrylic or a variety of other transparent materials(e.g., plastic). The aperture 5122 and window pane 5124 can assume avariety of shapes, but in some embodiments they are circular orelliptical. The window 5121 can be attached to the housing 5102 by aplurality of fasteners such as bolts 5126. In FIG. 12 , only two bolts5126 are shown in the cross-sectional view, but in some embodiments,additional bolts can be positioned along the edges of the window 5121.In some embodiments, the window 5121 can include a flange 5123 formounting the window. The flange 5123 may have a plurality of throughholes through which fasteners (e.g., bolts 5126) can pass to secure thewindow 5121 to the housing 5102. A seal 5128 (e.g., an O-ring) can bepositioned between the housing 5102 and the window 5121 (e.g., theflange 5123). The bolts 5126 can be tightened, causing the O-ring 5128to be compressed between the housing 5102 and the window 5121. In someembodiments, the O-ring 5128 produces a hermetic seal. Other approachescan be used to fasten the window 5121 to the housing 5102. For example,the window 5121 can be disposed in recess on the outer or inner surfaceof the housing 5102, or can be embedded into the housing 5102, or can bemounted onto the inside of the housing 5102. The window 5121 can besecured to the housing 5102 by an adhesive, epoxy, or cement.

Although the embodiment shown in FIG. 12 shows a single output window,multiple output windows can be used. For example, each beam of light5120A-5120D can exit the interior chamber 5304 via a respective outputwindow. In some embodiments, it is desirable that as much as possible ofat least the inner surface area of the housing 5102 comprise thethermally conductive material, to better achieve temperature uniformity.Accordingly, the output windows can be separated by thermally conductivematerial and can cover only as much area as necessary to allow lightbeams 5120A-5120D to leave the interior chamber 5104. However, in someembodiments a single output window is easier and less expensive toconstruct.

In some embodiments, the optical elements (e.g., lenses or lens) thatproduce the light beams 5120A-5120D can be formed as part of the outputwindow (or windows). For example, the window pane 5124 can include atleast one curved surface to produce optical power, which can beconfigured to produce the plurality of light beams 5120A-5120D having adesired shape and/or size. The window pane 5124 can comprise a lensarray such as a microlens array, and can be anamorphic as discussedabove.

The optical system 5100 can include a flow cell connector 5130 that isattached to the housing, and the flow cell connector 5130 is configuredto secure a flow cell 5132 so that it intersects the beams of light5120A-5120D. In some embodiments, the flow cell connector 5130 canpermanently attach the flow cell 5132 to the housing 5102. However, insome embodiments, the flow cell connector 5130 can allow the flow cell5132 to be removably attached to the housing 5102. In some embodiments,the flow cell connector 5130 can be compatible with multiple typesand/or sizes of flow cells. For example, the flow cell connector caninclude a clip, a friction or pressure fit coupling, a threaded portionconfigured to receive a corresponding threaded portion of the flow cell5132, or a variety of other connectors known in the art or yet to bedevised. The flow cell 5132 can be a capillary flow cell, and at leastpart of the flow cell can comprise a transparent material (e.g., glass)that allows the light beams 5120A-5120D to enter the flow cell 5132 andinteract with a sample fluid contained within the flow cell 5132. In oneembodiment, the flow cell 5132 can be a thin hollow tube, forming a flowpath that has a diameter of about 10 microns. Other flow cell typesand/or sizes can be used, and the flow cell 5132 can be orienteddifferently than as shown in FIG. 12 . In some embodiments, the beams oflight 5120A-5120D strike the flow cell over areas centered about 110 to140 microns apart from each other, and in some embodiments, 125 micronsapart from each other. For some forms of optical measurements, it isdesirable for the laser light to strike the flow cell at specificlocations (e.g., areas spaced about 125 microns apart). In someembodiments, the optical system 5100 mounts the optical fibers toautomatically direct the light from the laser light sources 5112A-5112Dto the desired locations of the flow cell 5132 without requiring theuser to manipulate any mirrors or wavelength selective elements such asdichroic mirrors or optical elements.

The optical system 5100 can be compatible with various types of optical(e.g., spectroscopic) analysis. For example, for laser-inducedfluorescence spectroscopy, a fluorescent dye designed to bond with ananalyte can be introduced into the fluid sample. When the fluid samplepasses through the beams of light 5120A-5120D, the fluorescent dyeabsorbs photons and emits photons that have a longer wavelength (lessenergy). By using photodetectors such as a photomultiplier tube (PMT)(not shown) to measure the amount of light that is emitted, the presenceor concentration of the analyte in the sample fluid can be measured. Forabsorption spectroscopy, photodetectors (not shown) can be positioned onthe side of the flow cell 5132 opposite the housing 5102 to determiningthe amount of light that is absorbed by the fluid sample. The opticalsystem 5100 can also be compatible with other types of opticalmeasurements or spectroscopic analysis.

FIG. 14 schematically shows an embodiment of an optical system 5300 thatcan be used to direct light for optical measurements (e.g.,laser-induced fluorescence and spectroscopic analysis). The opticalsystem 5300 is similar to optical system 5100 in some aspects, andsimilar elements are labeled with the same reference numerals used inFIG. 12 except that the numbers are increased by 200. The optical system5300 can include a flow cell connector 5330 that comprises a thermallyconductive auxiliary sample housing 5350 which encloses an interiorchamber 5352. The flow cell connector 5330 can be configured to secure aflow cell 5332 so that it passes through the interior chamber 5352. Forexample, the sample housing 5350 can include two apertures 5354, 5356and two flexible seals 5358, 5360, so that the flow cell 5332 can beslidably inserted through the apertures 5354, 5356 and held in place byfriction against the flexible seals 5358, 5360. Alternatively, thesample housing 5350 can include a door allowing the sample housing 5350to be opened and the flow cell 5332 to be placed inside. In variousembodiments where the interior chamber 5352 of sample housing 5350 ishermetically sealed with respect to interior chamber 5304 of the mainhousing 5302, the interior chamber 5352 of the sample housing 5350 canbe exposed to ambient air without exposing the components containedwithin interior chamber 5304 of the main housing 5302. Accordingly, theinterior chamber 5352 of the sample housing 5350 can be exposed toambient air when flow cell 5332 is removed and the seals 5358, 5360 maybe excluded in some embodiments.

In some embodiments, the sample housing 5350 can be integrally formed aspart of the main housing 5302 or can be thermally coupled to the mainhousing 5302 so that the thermoelectric controller 5306 regulates thetemperature within the interior chamber 5352 of the sample housing 5350as well as the interior chamber 5304 of the main housing 5302. In someapplications it may be desirable to maintain the internal chamber 5352of the sample housing 5352 enclosing the flow cell at a differenttemperature than the internal chamber 5304 of the main housing 5302,such as when a fluid sample is used that should be maintained at adifferent temperature than the interior chamber 5304 of the main housing5302. Accordingly, in some embodiments, a second thermoelectriccontroller (not shown) can be thermally coupled to the sample housing5350 and an insulating layer (not shown) can be positioned at thetransition between the main housing 5302 and the sample housing 5350 sothat the internal chamber 5352 of the sample housing 5350 can bemaintained at a different temperature than the interior chamber 5304 ofthe main housing 5302.

The optical system 5300 can include a second output window fortransmitting light out of the internal chamber 5352 of the samplehousing 5350. The second output window can be similar to the outputwindow described above, and cover an aperture 5362 covered with atransparent window pane 5364. The transparent window pane 5364 can beattached to the housing 5350 by bolts 5366 and sealed by a seal 5368. Insome embodiments, the interior chamber 5352 of the sample housing 5350is not hermetically sealed and the seal 5368 can therefore be anon-hermetic seal or can be omitted altogether.

FIG. 15 schematically shows an embodiment of an optical system 5400 thatcan be used to direct light for optical measurements such aslaser-induce fluorescence and spectroscopic analysis. The optical system5400 is similar to optical systems 5100 and 5300 in some aspects, andsimilar elements are labeled with the same reference numerals used inFIGS. 12 and 14 except that the numbers are increased by 200 and 100respectively. Optical system 5400 can include a flow cell connector 5430that attaches a flow cell 5432 to the housing 5402 so that the flow cell5432 passes through the housing 5402. For example, the housing 5402 cancomprise two apertures 5454, 5456 and two flexible seals 5458, 5460, sothat the flow cell 5432 can be slidably inserted through the apertures5454, 5456 and held in place by friction against the flexible seals5458, 5460. Alternatively, the housing 5402 can include a door allowingthe housing 5402 to be opened and the flow cell 5432 to be placedinside. In some embodiments, the interior chamber 5404 can be exposed toambient air when flow cell 5432 is removed and the seals 5458, 5460 canbe non-hermetic seals. Also, the seal 468 can be a non-hermetic seal orcan be omitted altogether.

As discussed above, various embodiments of the optical systems describedherein can be used to perform a variety of optical measurements, suchas, for example laser-induced fluorescence (LIF) measurements.Embodiments disclosed herein, such as those using LIF, can be useful inbiological and/or chemical analysis. For example, LIF can be used in DNAsequencing applications, molecular diagnostic applications, confocalmicroscopy, or flow cytometry. FIG. 16 illustrates an embodiment of anoptical system 1600 configured to analyze a sample 1613, for exampleusing laser induced fluorescence. The optical system comprises a lightsource 1601 configured to direct light at one or more incidentwavelengths towards the sample 1613 along an incident path. Light fromthe light source 1601 can be focused onto a portion of the sample 1613using a lens 1611. In various implementations, the lens 1611 cancomprise a microscope objective lens. In some implementations, the lens1611 can comprise an infinity corrected objective lens. Other types offocusing lenses can also be used as the lens 1611. In some embodiments,the lens 1611 can include multiple lens elements. The light source 1601can comprise one or more lasers and/or one or more light emitting diodes(LEDs). The light source 1601 can be configured to emit light at one ormore incident wavelengths in a wavelength range between about 250 nm andabout 980 nm, or an values or ranges therebetween, in some examples. Theoptical power of the one or more incident wavelengths emitted from thelight source 1601 can be between about 1 mW and about 10,000 mW, or anyvalues or ranges there between, in some example. Various types of laserscan be used, including continuous-wave (CW) lasers, quasi CW lasers,pulsed lasers, laser diodes, multi-mode lasers, etc. The sample 1613 maybe a biological or a chemical sample, which can be tagged with one ormore fluorescent dyes that absorb the light at the one or more incidentwavelengths and emit fluorescence radiation at one or more fluorescentwavelengths. The fluorescence radiation at the one more fluorescentwavelengths emitted by the sample 1613 is directed along an emissionpath and imaged by an imaging device 1603, such as, for example, acamera.

In various implementations, a portion of the incident path and theemission path can be combined using a beam combiner 1609, such as, forexample a dichroic mirror. The beam combiner 1609 can be configured toreflect incident light from the light source 1601 towards the sample1613 and can transmit fluorescence radiation at the one or morefluorescent wavelengths towards the imaging device 1603. In variousimplementations, a wavelength selective filter 1602 can be disposed inthe incident path to transmit light at one or more selected wavelengths.The wavelength filter 1602 can be disposed in a filter wheel or otherfilter switcher comprising a plurality of wavelength selective filtersconfigured to transmit light at one or more different wavelengths. Thefilter 1602 can be used to control the wavelength of light thatilluminates the sample. For example, the light source 1601 can outputblue, green, and red light, and the filter 1602 can attenuate the redand green light while permitting the blue light to be directed to thesample. The filter 1602 can be selectively change (e.g., using thefilter wheel or other filter selector) in order to change the wavelengthof light that is directed to the sample 1613 (while using the same lightsource 1601). Various different colors of lasers and filters can beused, such as depending on the florescent dyes that are used. A secondwavelength selective filter 1607 can be disposed in the emission path totransmit fluorescence radiation at one or more selected fluorescentwavelengths. The second wavelength filter 1607 can also be disposed in afilter wheel or other filter selector comprising a plurality ofwavelength selective filters configured to transmit light at one or moredifferent wavelengths. The filter 1607 can be selectively changed inorder to control the wavelength of light that reaches the camera 1603.The emission path can comprise one or more optical lenses 1605. In someimplementations, the one or more optical lenses 1605 can be a tube lens.The one or more optical lenses 1605 can focus the light onto the camera1603.

FIG. 17 illustrates another embodiment of an optical system 1700configured to analyze biological and/or chemical samples usinglaser-induced fluorescence. Various aspects of the optical systemdepicted in FIG. 17 can be similar to the optical system 1600 depictedin FIG. 16 . The optical system comprises a light source 1701 (e.g., a4-channel light source) configured to emit incident light that isfocused onto a sample using a lens 1711 (e.g., a microscope objectivelens, an infinity-corrected objective lens, etc.). The lens 1711 can bemounted on a translational stage 1719 configured to adjust the positionof the focus with respect to the sample. The optical system can comprisean autofocus system 1717 configured to focus the incident light onto thesample. Fluorescence radiation emitted from the sample is split alongtwo imaging paths and imaged by two imaging devices 1803 (e.g., camera).A filter wheel 1707 or other filter switcher can be disposed in one ofthe two imaging paths to select one or more fluorescent wavelengths.Many variations are possible. For example, the filter 1707 can beomitted in some embodiments. Multiple cameras can be used, and multipledichroic mirrors can be used to direct particular wavelengths of lightto the cameras.

Referring to FIG. 16 , the sample 1613 can be contained in a flow cell.An example flow cell 1830 is depicted in FIG. 18 . The flow cell 1830can comprise an array of tiles 1833. Different tiles in the array cancontain different samples to be analyzed. The samples can include DNA,DNA clusters, bacteria, chemical formulations, etc. The optical system1600 (and/or the optical system 1700) can be configured to analyze thedifferent samples contained in the array of tiles by causing relativemovement between the flow cell 1830 and the optical system 1600. Forexample, the lens 1611 (or the lens 1711) or the flow cell 1830 can bemoved in a plane normal to the optical axis of the lens 1611 (or thelens 1711) such that light from the light source 1601 (or the lightsource 1701) at the one or more incident wavelengths is focused ontodifferent tiles of the tile array at different times. An actuator canmove the flow cell and/or optical system relative to each other, such asin two dimensions, such as using an XY stage. To efficiently excite onlythe sample contained in the tile onto which the light from the lightsource 1601 (or the light source 1701) at the one or more incidentwavelengths is focused, it is desirable that the spot size of thefocused light is matched to the size of the tile and that the intensityof light is substantially uniform across the area of the tile. Forexample, as shown in the inset of FIG. 18 , the illumination profile issubstantially uniform across the surface of the tile 1833. As usedherein, intensity variation less than or equal to about 20% from anaverage intensity can be considered to be substantially uniform. Asillustrated in FIG. 18 , the illumination profile can be substantiallyuniform across a substantial portion (e.g., greater than or equal toabout 50%) of the length and the breadth of tile 1833. For example,light intensity along the axis 1835 parallel to the breadth of the tile1833 can be substantially uniform (e.g., intensity variation less thanor equal to about 20% from an average intensity) over at least 50% ofthe breadth of the tile 1833. The light intensity along the axis 1837parallel to the length of the tile 1833 can be substantially uniform(e.g., intensity variation less than or equal to about 20% from anaverage intensity) over at least 50% of the length of the tile 1833. Thelight intensity across a substantial portion of the breadth of the tile1833 can be approximately equal to the light intensity across asubstantial portion of the length of the tile 1833. Without any loss ofgenerality, the variation of light intensity across the length and thebreadth can be substantially flat for a substantial portion of thelength and breadth and rapidly fall off near the edges of the tile 1833to advantageously allow uniform illumination across the surface of thetile 1833 without exciting the samples in adjacent tiles. For example,the intensity of any side lobes can be less than or equal to 1%. In thismanner, it can be accomplished that only the fluorescent dyes tagged tothe sample contained in the tile 1833 are excited by the incident lightand fluorescent dyes tagged to different samples in adjacent tiles arenot excited by the incident light. Systems and methods of generatinglight with uniform intensity profile for laser-induced fluorescence arediscussed below. Many variations are possible. The light intensity canvary by less than or equal to about 2%, about 3%, about 5%, about 7%,about 10%, about 15%, about 20%, about 25%, about 30%, or any values orranges therebetween, across at least about 30%, about 40%, about 50%,about 60%, about 70%, about 80%, or about 90% of the area of the tile,or of the length and/or breadth of the tile, or any values or rangestherebetween. In some embodiments, a flat-top distribution of light canbe produced, as discussed herein. The light delivered to the flow cellcan have a light profile shape that generally conforms to the shape ofthe flow cell or frame 1833. In the illustrated embodiment, the frame1833 has a square shape, and the light delivered to the frame 1833 canhave a generally square shaped distribution (e.g., square with roundededges flat-top distribution). Other shapes of light distribution can beused, such as rectangular, circular, triangle-shaped, etc. The morelight intensity and/or covered area that is put into the sample, theshorter the exposure time can be for the camera, which can result infaster measurement times and more throughput. The improved illuminationembodiments disclosed herein can also provide improved image quality andmeasurement accuracy.

Without any loss of generality, intensity light from the light source1601 (or the light source 1701) comprising one or more lasers or LEDsneed not be uniform across the length and the breadth or area of thetile. For example, the variation in the intensity across the lengthand/or the breadth of the tile can be greater than or equal to about 20%of the average light intensity for the substantial portion of the lightarea. FIG. 19A depicts the intensity across the length extending alongaxis 1937 and across the breadth extending along axis 1935, for lightoutput by an example light source. The intensity profile on the left ofFIG. 19A corresponds to the variation of intensity across the breadthextending along axis 1935 and the intensity profile on the right of FIG.19A corresponds to the variation of intensity across the lengthextending along axis 1937. As noted from FIG. 19A, the variation ofintensity across the breadth extending along axis 1935 and across thelength extending along axis 1937 is not uniform and exhibits many peaksvalleys. In some embodiments, the light source 1601 or 1701 can use oneor more multi-mode lasers (e.g., laser diodes), which in some cases cancontribute to the non-uniformity of the light distribution. Multi-modelaser diodes can be advantageous for availability and cost reasons, andthe optical system can be configured to modify the light to increaseuniformity.

Using optical systems and methods described herein, the variation ofintensity across the breadth extending along axis 1935 and across thelength extending along axis 1937 can be configured to be substantiallyuniform as shown in FIG. 19B. For example, using optical systems andmethods described herein, the variation of intensity across the breadthextending along axis 1935 can be made substantially uniform across asubstantial portion of the breadth of the tile as shown in the intensityprofile on the left of FIG. 19B. Similarly, the variation of intensityacross the length extending along axis 1937 can be made substantiallyuniform across a substantial portion of the length of the tile as shownin the intensity profile on the right of FIG. 19B. As noted from theintensity profiles on the left and the right of FIG. 19B, the intensityprofile has a smooth flat top with steep sides to provide uniformillumination across the length and the breadth or area of tile whiledropping of quickly at the edges of the tile to reduce the chance ofexciting samples in neighboring tiles.

To achieve an intensity profile with a smooth flat top that falls offrapidly near the edges of the tile as shown in FIG. 19B, a beam shapingsystem can be disposed in the incident path. The beam shaping system cancomprise a focusing lens, a dynamic diffuser, and a micro-lens array, insome embodiments. FIG. 20 depicts the incident path of an embodiment ofan optical system 2000 configured to analyze samples (e.g., biologicaland/or chemical samples), such as using laser-induced fluorescence. Theoptical system 2000 can be an illumination system, such as forilluminating a sample (e.g., to induce fluorescence). The incident pathof the optical system 2000 comprises a plurality of laser diodes 2001 a,2001 b and 2001 c. The plurality of laser diodes 2000 a-2000 c can beconfigured to output laser light at red, green and blue wavelengths,although any suitable wavelengths, or any suitable number of lightsources (e.g., lasers) can be used. In various embodiments, theplurality of laser diodes 2001 a-2001 c can be further configured tooutput amplified spontaneous emission (ASE) along with laser light(e.g., at red, green and blue wavelengths). The optical system caninclude one or more lenses to modify light emitted by the one or morelasers 2001 a-c. For example, collimating lenses (e.g., aspheric lenses)can be used to collimate light from the lasers 2001 a-c. The opticalsystem can include one or more filters to modify light emitted by theone or more lasers 2001 a-c. For example, clean-up filters can be usedto attenuate some frequencies of light from the lasers. For example, thefilters can attenuate light at portions of the tails of laser light,while permitting light at the peaks of the laser light to passsubstantially unattenuated.

The laser light (e.g., at red, green and blue wavelengths) can becombined using beam combiners 2003 (e.g., which can include dichroicmirrors). The combined beam (e.g., having light at red, green and bluewavelengths) can be focused onto a dynamic diffuser 2007 using afocusing optical element (e.g., lens) 2005. In various implementations,the focusing lens 2005 can be either be a plano-convex or a focusingaspheric lens. Light output from the dynamic diffuser 2007 can becollimated using a collimating optical element (e.g., lens) 2009 andincident on a micro-lens array 2011.

Light output from the micro-lens array can be incident on the samplecontained in a flow cell 2030 via a dichroic mirror (e.g., a beamcombiner) 2013 and a focusing lens 2015. The dichroic mirror 2013 canoperate similar to the dichroic mirror 1609 of FIG. 16 . The dichroicmirror 2013 can reflect light from the light source (e.g., lasers 2001a-c) for illuminating a sample, and the dichroic mirror 2013 cantransmit light emitted from the sample (e.g., by fluorescence) forimaging. The flow cell 2030 can comprise an array of tiles as shown inFIG. 18 . The focusing lens 2015 can be a microscope objective (e.g.,which can have multiple lens elements, as shown). For example, thefocusing lens 2015 can be an objective lens having an effective focallength (EFL) of about 14.43 mm and a numerical aperture (NA) of 0.68,although various other configurations are possible (e.g., focal lengthbetween about 5 mm and about 25 mm, or values outside this range).

In various implementations, light at amber wavelength, for example, canbe combined with the laser light (e.g., of red, green and bluewavelengths) either before the focusing lens 2005 of the beam shapingsystem or collimating lens 2009 using another beam combiner. In someembodiments, one or more LED light sources 2001 d can be used. Acollimating optical element, such as a collimating lens (e.g.,aspherical lens) can collimate light from the LED. In some cases afilter 2017, such as a band pass filter, can attenuate some wavelengthsof light, while passing at least some wavelengths of light output by theLED. Light from the one or more LEDs can be combined (e.g., with thelaser light) using one or more beam combiners 2019 (e.g., one or moredichroic mirrors, as shown in FIG. 20 ). In some embodiments, the LED2001 d, filter 2017, and/or dichroic mirror 2019 can be omitted. In someembodiments, light from the diffuser 2007 can be reflected by thedichroic mirror 2019 to the micro-lens array 2013. In some embodiments,LED light sources similar to 2001 d can be used in place of lasers 2001a-c. Many variations are possible.

The micro-lens array can modify the distribution of light, such as toincrease uniformity. By way of example, various lenslets (e.g., each ofa plurality of lenslets) of the micro-lens array 2011 can sample theincident illumination profile. Each sample can be imaged onto the fieldof view (FOV) at the plane of the flow cell 2030 with the focusing lens2015. The samples from the various lenslets (e.g., each lenslet) ofmicro-lens array 2011 can add together to generate a flat-top profile inthe plane of the flow cell 2030, e.g., similar to the intensity profilesdepicted in FIG. 19B.

The distance between the focusing lens 2005 and the dynamic diffuser2007 can be adjusted to achieve a spot size sufficiently small toilluminate a target (e.g., a desired region of the flow cell 2030) andhave sufficiently high intensity to excite fluorescence in the sample.For example, the distance between the focusing lens 2005 and the dynamicdiffuser 2007 can be adjusted such that a size of the focused spot onthe desired region of the flow cell 2030 is less than or equal to thesize of the desired region of the flow cell 2030 and the focused spothas a sufficiently high intensity to excite fluorescence in the sample.As another example, consider that the desired region of the flow cell2030 is a square or a rectangle shaped region having a length ‘1’ and abreadth ‘b’. The distance between the focusing lens 2005 and the dynamicdiffuser 2007 can be adjusted such that focused spot has a square or arectangle shape having a length and breadth less than or equal to thelength ‘1’ and breadth ‘b’ of the selected region of the flow cell 2030and has a sufficiently high intensity to excite fluorescence in thesample. In some implementations, the distance between the focusing lens2005 and the dynamic diffuser 2007 can be adjusted such that focusedspot has a circular shape having a diameter less than or equal to thelength ‘1’ and/or breadth ‘b’ of the selected region of the flow cell2030 and has a sufficiently high intensity to excite fluorescence in thesample. In some implementations, the distance between the focusing lens2005 and the dynamic diffuser 2007 can be adjusted such that focusedspot has an elliptical shape wherein a length of the major axis and theminor axis is less than or equal to the length ‘1’ and/or breadth ‘b’ ofthe selected region of the flow cell 2030 and has a sufficiently highintensity to excite fluorescence in the sample. Various other shapes forthe focused spot are contemplated herein.

The dynamic diffuser 2007 can comprise an optical diffuser coupled to amechanical actuator. The dynamic diffuser 2007 can move (e.g., vibrate).The mechanical actuator can be configured to move the optical diffuseralong one or more directions in the plane of the diffuser. For example,the diffuser 2007 can move (e.g., vibrate) in one dimension (e.g., aback and forth motion). The diffuser 2007 can move in two-dimensions(e.g., a circular motion) while the distance between the diffuser 2007and the focusing optical element 2005 remains substantially constant. Insome cases, the diffuser can be configured to move along one or moredirections that are substantially orthogonal to a normal to the plane ofthe diffuser. Additionally, the diffuser can be configured to move in adirection substantially orthogonal to the beam such that the beam cansample different regions of the diffuser at different times. Thediffuser can be configured to move at a frequency of a few 100 Hz. Invarious implementations, the mechanical actuator can be configured tovibrate the optical diffuser e.g., along one or more directions in theplane of the diffuser. For the laser beam having a generally ellipticalspot (e.g., having a width along a major axis that is longer than awidth along a minor axis), the mechanical actuator can be configured tomove/vibrate the optical diffuser along a direction that issubstantially parallel (e.g., within about 1 degree, about 2 degrees,about 5 degrees, about 10 degrees, or any values or ranges therebetween,although other values can be used) to the direction of the major axis ofthe generally elliptical laser beam. The diffuser can move more in thedirection of the major axis than in the direction of the minor axis(e.g., which can be orthogonal to the major axis). Movement along themajor axis can decrease the area of the diffuser that is illuminated atonly sometimes as the diffuser moves, as compared to movement along theminor axis. This can promote uniformity of the light, because movementalong the major axis causes more of the light to merely move around onthe portions of the diffuser that were already illuminated. Moving(e.g., vibrating) the diffuser can advantageously reduce the speckle(e.g., which can result from the laser light), and can help achieve thesmooth (e.g., flat-top) illumination profile.

The optical diffuser can increase etendue (or divergence) of the laserlight. Accordingly, focusing the laser light using the focusing lens2005 can advantageously help to maintain the etendue of the laser beamafter the beam shaping element below a threshold etendue value. Forexample, the divergence of the laser beam after the dynamic diffuser2007 can be between about 4 degrees and about 30 degrees, or any valuesor ranges therebetween, although other configurations are also possible.Without being bound by any theory, the smaller the spot size of thelight focused by the focusing optical element 2005 onto the diffuser2007, the more the etendue of the light can be maintained. Focusing thelight onto a relatively small spot size on the diffuser 2007 can producelight having lower etendue, as compared to focusing light onto arelatively large spot size on the diffuser 2007. In various embodiments,the maximum spot size of the light focused by the focusing element 2005at the diffuser can be determined based on the etendue of the opticalelement receiving the light from the diffuser 2007. For example, asdiscussed below with reference to FIG. 22 , for optical fiber basedapplications, the maximum spot size at the diffuser 2007 can depend onthe core diameter and numerical aperture of the optical fiber into whichthe light output from the diffuser is coupled. As another example, foroptical fiber based applications, the spot size at the diffuser 2007 canhave a size that is sufficiently large to generate diffused light withRMS noise less than about 10% and that is small enough to be coupledinto an optical fiber using compact coupling optical systems.

To minimize the etendue of the laser light after the dynamic diffuser2007, it is advantageous to adjust the distance between the focusinglens 2005 and the dynamic diffuser 2007 such that the laser light isfocused onto the dynamic diffuser 2007 and the spot size of the laserlight at the output of the dynamic diffuser 2007 is minimized. However,it was discovered that minimizing the etendue by minimizing the spotsize of the laser light at the output of the dynamic diffuser 2007 candisadvantageously increase the intensity noise of the output light. Thisis illustrated in FIG. 21 which shows the variation of the beam diameterin the horizontal direction (represented by curve 2105) and the verticaldirection (represented by curve 2110) in the plane of the dynamicdiffuser 2007 as a function of the distance between the focusing lens2005 and the dynamic diffuser 2007. The corresponding intensity noise asa function of the distance between the focusing lens 2005 and thedynamic diffuser 2007 is represented by curve 2115. Without any loss ofgenerality, the intensity noise is measured as the percentage variationin the root mean square (RMS) noise with respect to an averageintensity. For example, RMS noise is obtained by calculating the ratioof the variation in time in the RMS value of the AC component of theelectrical signal generated by the laser light in a photodiode to anaverage value of the DC component of the electrical signal generated bythe laser light in the photodiode. As observed from FIG. 21 , in thisexample, the smallest beam diameter in the horizontal direction(represented by curve 2105) is obtained when the distance between thefocusing lens 2005 and the dynamic diffuser 2007 is about 5.75 mm andthe smallest beam diameter in the vertical direction (represented bycurve 2110) is obtained when the distance between the focusing lens 2005and the dynamic diffuser 2007 is about 5.2 mm. The distance between thefocusing lens 2005 and the dynamic diffuser 2007 that results in thesmallest beam diameter in the horizontal/vertical direction cancorrespond to the focal distance of the focusing lens 2005. Theintensity noise has a maximum value when the distance between thefocusing lens 2005 and the dynamic diffuser 2007 is about 5.2 mm.Accordingly, it can be concluded that when the distance between thefocusing lens 2005 and the dynamic diffuser 2007 is equal to the focaldistance of the focusing lens 2005 or is within ±10% of the focaldistance, the spot size of the laser light at the output of the dynamicdiffuser 2007 has the smallest beam diameter in the horizontal/verticaldirection and an increased intensity noise. The increase in theintensity noise can be at least partially attributed to the variation intime in the transmissivity through the diffuser as the dynamic diffuser2007 is moved/vibrated. As the dynamic diffuser 2007 is moved/vibratedthe laser beam from the focusing lens 2005 is incident at differentportions of the dynamic diffuser 2007 which can have differenttransmissivities. This can result in a variation in the intensity oflight transmitted through the diffuser which manifests as intensitynoise. From FIG. 21 it is observed that as the dynamic diffuser 2007 isdisplaced toward or away from the focal plane of the focusing lens 2005(e.g., by a distance that is greater than about 10% of the focaldistance of the focusing lens 2005), then the intensity noise decreases.In other words, as the light output from the focusing lens 2005 isfocused above or below the plane of the dynamic diffuser 2007 theintensity noise decreases. Without being bound by theory, it is believedthat as the spot size of the light on the diffuser is increased and thelight ‘samples’ a larger area of the diffuser, then the properties ofthe diffuser average across the illuminated area average out so thatmovement of the illuminated spot on the diffuser (e.g., by vibration ofthe diffuser) does not make a significant different in the averagedproperties of the diffuser. In contrast, a very small spot size canexperience more significant changes as it ‘samples’ different portionsof the diffuser. Accordingly, it can be advantageous to increase thespot size of the light on the diffuser 2007. However, a larger spot sizeon the diffuser can resulted in increased etendue or other undesiredoptical qualities. Accordingly, a balance can be performed betweenproducing a sufficiently large spot size to have low noise, while alsoproducing a sufficiently small spot size to have the desired opticalqualities (e.g., low etendue). However, focusing the light output fromthe focusing lens 2005 above or below the plane of the dynamic diffuser2007 can result in an increase in the beam diameter horizontal/verticaldirection and/or an increase the spot size of the laser beam at theoutput of the dynamic diffuser 2007.

Without being bound by any theory, the amount of intensity noise in thelight at the output of the diffuser can depend on the size anddistribution of the diffusing elements of the dynamic diffuser 2007. Forexample, an implementation of a dynamic diffuser 2007 having regionswith higher concentration of the diffusing elements interspersed withregions having lower concentration of the diffusing elements can havehigher intensity noise as compared to an implementation of a dynamicdiffuser 2007 in which the diffusing elements are uniformly dispersed.Accordingly, the spot size of the light focused on the implementation ofthe dynamic diffuser 2007 in which the diffusing elements are uniformlydispersed can be smaller (e.g., low etendue) than the spot size of thelight focused on the implementation of the dynamic diffuser 2007 havingregions with higher concentration of the diffusing elements interspersedwith regions having lower concentration of the diffusing elements toachieve a similar amount of intensity noise. Thus, based on the physicaland optical characteristics of the dynamic diffuser 2007, the spot sizeof the light focused by the focusing element 2005 on the dynamicdiffuser 2007 can be selected to achieve a desired intensity noise.

In some embodiments, the optical system can be configured so that thefocusing optical element 2005 produces a spot of light on the diffuser2007 that has a width (e.g., diameter) larger than about 20 microns,about 25 microns, about 30 microns, about 50 microns, about 75 microns,about 100 microns, about 150 microns, about 200 microns, about 250microns, about 300 microns, or more (as shown in FIG. 21 ), or anyvalues therebetween, or any ranges bounded therebetween. In someembodiments, the optical system can be configured so that the focusingoptical element 2005 produces a spot of light on the diffuser 2007 thathas an area larger than about 200 square microns, about 300 squaremicrons, about 500 square microns, about 750 square microns, about 1,000square microns, about 1,250 square microns, about 1,500 square microns,about 1,750 square microns, about 2,000 square microns, about 2,500square microns, about 3,000 square microns, about 4,000 square microns,about 5,000 square microns, about 7,500 square microns, about 10,000square microns, about 15,000 square microns, about 20,000 squaremicrons, about 25,000 square microns, about 50,000 square microns, about75,000 square microns, about 100,000 square microns, about 150,000square microns, or more, or any values therebetween, or any rangesbounded therein. In some embodiments, the diffuser 2007 can be spacedaway from the focus or focal plane of the focusing optical element 2005so that defocused light forms the spot of light on the diffuser. In someembodiments, the focusing optical element 2005 or other features of theoptical system can be configured so that the spot of light produced atthe focus or focal plane of the focusing optical element has a size thatis sufficiently large to reduce light intensity noise, as discussedherein. Various embodiments described herein are configured such thatthe spot size of the light output from the focusing element 2005 at thediffuser 2007 has a spot size that is large enough to reduce intensitynoise (e.g., spot size large enough such that the light at the output ofthe diffuser has a RMS noise less than 10%) and small enough to matchthe etendue of the optical element that receives the light output fromthe diffuser. For example, for some applications, the maximum spot sizeof the light at the diffuser can be 200 μm×200 μm.

Without any loss of generality, the distance between the focusing lens2005 and the dynamic diffuser 2007 can be adjusted to any distancegreater than or less than the focal distance of the focusing lens 2005that generates a laser spot having a size that matches or substantiallymatches (e.g., within ±10%, within ±5%, within ±2%, etc.) of the size ofthe region of the flow cell 2030 that is desired to be illuminated suchthat the RMS noise is less than or equal to about 10%. For example, thedistance between the focusing lens 2005 and the dynamic diffuser 2007can be adjusted to any distance greater than or less than the focaldistance of the focusing lens 2005 that generates a laser spot having asize that matches or substantially matches (e.g., within ±10%, within±5%, within ±2%, etc.) the size of the desired region of the flow cell2030 such that the RMS noise is less than or equal to about 8%, lessthan or equal to about 5%, less than or equal to about 3%, less than orequal to about 2%, less than or equal to about 1.5%, less than or equalto about 1%, or less (as shown in FIG. 21 ), or any value in a rangedefined by any of these values. The term “substantially” is s generalterm as used herein, and when used in conjunction with a number or arange forms a phrase that will be readily understood by a person ofordinary skill in the art. For example, is readily understood that suchlanguage will include a number or range were little difference isdiscernible or matters. For example, the term substantially can meanwithin 20% of the number or the range. In various implementations, thedistance between the focusing lens 2005 and the dynamic diffuser 2007can be adjusted to any distance greater than or less than the focaldistance of the focusing lens 2005 that generates a laser spot having asize that is greater than or equal to about 10% of the size of thedesired region of the flow cell 2030 such that the RMS noise is lessthan or equal to about 10%. For example, the distance between thefocusing lens 2005 and the dynamic diffuser 2007 can be adjusted to anydistance greater than or less than the focal distance of the focusinglens 2005 that generates a laser spot having a size that is greater thanor equal to about 20% of the size of the desired region of the flow cell2030, greater than or equal to about 30% of the size of the desiredregion of the flow cell 2030, greater than or equal to about 40% of thesize of the desired region of the flow cell 2030, greater than or equalto about 50% of the size of the desired region of the flow cell 2030,greater than or equal to about 60% of the size of the desired region ofthe flow cell 2030, greater than or equal to about 70% of the size ofthe desired region of the flow cell 2030, greater than or equal to about80% of the size of the desired region of the flow cell 2030, greaterthan or equal to about 90% of the size of the desired region of the flowcell 2030, and less than or equal to the size of the desired region ofthe flow cell 2030. Although various embodiments are discussed inconnection with the spot size relative to the size of the desired regionof the flow cell, any suitable target can be used instead of a flowcell. For example, the embodiments discussed herein can be modified tohave spot size relative to an optical fiber end or coupler, or any othersuitable target.

In various implementations, the dynamic diffuser 2007 can be displacedtoward or away from the focal plane of the focusing lens 2005 by adistance that is greater than about 12% of the focal distance of thefocusing lens 2005, a distance that is greater than about 15% of thefocal distance of the focusing lens 2005, a distance that is greaterthan about 20% of the focal distance of the focusing lens 2005, adistance that is greater than about 25% of the focal distance of thefocusing lens 2005, a distance that is greater than about 30% of thefocal distance of the focusing lens 2005, a distance that is greaterthan about 35% of the focal distance of the focusing lens 2005, adistance that is greater than about 40% of the focal distance of thefocusing lens 2005, a distance that is greater than about 45% of thefocal distance of the focusing lens 2005, and a distance that is lessthan about 50% of the focal distance of the focusing lens 2005, or anyvalues or ranges therebetween, such as to reduce the RMS noisepercentage and/or to generate a laser or light spot having a size thatis greater than or equal to about 10% of the size of the target (e.g.,the desired region of the flow cell 2030) and less than or equal to thesize of the target (e.g., the desired region of the flow cell 2030). Itis noted that in the various implementations discussed above, theintensity across the target (e.g., the surface of the desired region ofthe flow cell 2030) is substantially uniform (e.g., within ±20% of anaverage light intensity) for a substantial portion thereof.

Although various embodiments are discussed in connection with directinglight to a sample (e.g., a flow cell), various other targets for thelight can be used, such as for fiber couplings. The beam shaping systemdescribed above can also be integrated with fiber-coupled illuminationsources. FIG. 22 illustrates an embodiment of a fiber coupledilluminator comprising two laser diodes 2201 a and 2201 b respectivelycoupled to a collimator 2203 a and 2203 b. The light output from the twolaser diodes 2201 a and 2201 b is combined with a beam combiner 2205(e.g., a dichroic mirror) and focused by a focusing lens 2207. A dynamicdiffuser 2209 can be positioned away from the focal plane of thefocusing lens 2207 such that the combined laser beam from the two laserdiodes 2201 a and 2201 b is focused above or below the plane of thedynamic diffuser 2209. Light output from the dynamic diffuser 2209 iscoupled into a multimode fiber 2213, such as using coupling lens system2211. In various implementations, the multimode fiber 2213 can comprisea square or a rectangle shaped core such that intensity profile of thelight output from the multimode fiber has a smooth flat top as shown inFIG. 19B. In various implementations, the multimode fiber can have a 300micron core. In some implementations, a micro-lens array can be coupledat the output of the multimode fiber as discussed above with referenceto FIG. 20 .

As discussed above, the dynamic diffuser 2209 can be displaced toward oraway from the focal plane of the focusing lens 2207 by a distance thatis greater than about 10% of the focal distance of the focusing lens2207, a distance that is greater than about 12% of the focal distance ofthe focusing lens 2207, a distance that is greater than about 15% of thefocal distance of the focusing lens 2207, a distance that is greaterthan about 20% of the focal distance of the focusing lens 2207, adistance that is greater than about 25% of the focal distance of thefocusing lens 2207, a distance that is greater than about 30% of thefocal distance of the focusing lens 2207, a distance that is greaterthan about 35% of the focal distance of the focusing lens 2207, adistance that is greater than about 40% of the focal distance of thefocusing lens 2207, a distance that is greater than about 45% of thefocal distance of the focusing lens 2207, and a distance that is lessthan about 50% of the focal distance of the focusing lens 2207, or anyvalues or ranges therebetween, such as generate a laser or light spothaving a size that is greater than or equal to about 10% of the size ofa region to be illuminated and less than or equal to the size of theregion to be illuminated. The region to be illuminated can be a portion(e.g., a tile) of a flow cell or a portion of an optical fiber (e.g.,single mode or multi-mode fiber), or coupling. The distance between thedynamic diffuser 2209 and the focusing lens 2207 can be selected suchthat the RMS noise percentage has a value less than about 10% (e.g., avalue between about 1% or about 2% and about 10%). As discussed above,the intensity across a substantial portion of the region to beilluminated can be substantially uniform (e.g., within ±20% of anaverage light intensity).

FIGS. 23A-23E show the variation in the spot size at the output of thedynamic diffuser 2209 as the distance between the focusing lens 2207 andthe dynamic diffuser 2209 is increased from a value less than the focaldistance to a value greater than the focal distance. FIG. 23Aillustrates a laser spot having a size of about 360 μm×200 μm at theoutput of the dynamic diffuser 2209 when the dynamic diffuser 2209 isdisposed several millimeters above the focal plane of the focusing lens2207. FIG. 23B illustrates a laser spot having a size of about 200μm×170 μm at the output of the dynamic diffuser 2209 when the dynamicdiffuser 2209 is moved closer toward the focal plane of the focusinglens 2207. FIG. 23C illustrates a laser spot having a size of about 120μm×140 μm at the output of the dynamic diffuser 2209 when the dynamicdiffuser 2209 is moved further closer toward the focal plane of thefocusing lens 2207. FIG. 23D illustrates a laser spot having a size ofabout 2 μm×130 μm at the output of the dynamic diffuser 2209 when thedynamic diffuser 2209 is disposed at the focal plane of the focusinglens 2207. FIG. 23E illustrates a laser spot having a size of about 120μm×140 μm at the output of the dynamic diffuser 2209 when the dynamicdiffuser 2209 is moved away from the focal plane of the focusing lens2207 such that the focal plane of the focusing lens 2207 is above theplane of the dynamic diffuser 2209.

FIG. 24 is a flow diagram illustrating a process 2400 of illuminating atarget. At block 2405 the process 2400 includes receiving light from oneor more lasers at one or more beam combiners. Each of the one or morebeam combiners is positioned and aligned to receive light from acorresponding one of the one or more lasers. In some embodiments, theone or more beam combiners can be dichroic beam combiners. At block2410, the process 2400 includes combining, by the one or more beamcombiners, the received light and directing the received light along acommon path. The beam combiners of an illumination system can bepositioned in the system in various ways. For example, FIG. 20 and thecorresponding description illustrates an embodiment having beamcombiners directing light from a plurality of lasers along a common path(or the light is at least substantially aligned along a common path).

At block 2415, the process 2400 receives the light propagating along thecommon path at a focusing optical element and focuses the light. Forexample, the focusing optical element focuses the light towards a focuspoint (or focal point). The focusing optical element can be, in someembodiments, focusing lens 2005 (FIG. 20 ). At block 2420 of the process2400, the focused light is received at a diffuser. The diffuser can bepositioned such that it is not at the focus point. That is, the diffuseris positioned at a location spaced apart from the focus point. In someembodiments, the diffuser is positioned between the focusing opticalelement and the focus point such that light propagating from thefocusing optical element converging to the focus point is incident onthe diffuser. In some embodiments, the diffuser is positioned such thatthe focus point is between the focusing optical element and the diffusersuch that light propagating from the focusing optical element haspropagated past the focus point and when the light is incident on thediffuser. For example, such that light propagating past the focus pointis diverging when it is incident on the diffuser. At block 2425 ofprocess 2400, the light is diffused by the diffuser, and lightpropagating from the diffuser is diffused light. In various embodiments,the diffuser is moved, or vibrated diffusing the light, at least in partby vibrating the diffuser, and light from the diffuser is output asdiffused light. In some embodiments, vibrating the diffuser comprisesmoving the diffuser in a two-dimensional motion. In some embodiments,the two-dimensional motion is substantially orthogonal to an opticalaxis of the focusing optical element. In some embodiments, vibrating thediffuser comprises moving the diffuser in a plane. In some embodiments,vibrating the diffuser comprises moving the diffuser in a circularmotion. In some embodiments, vibrating the diffuser comprises moving thediffuser in a linear motion. At block 2530 of process 2400, light fromthe diffuser is received by a collimating optical element, which outputscollimated light. Collimating lens 2009 in FIG. 16 is one example of adevice for performing this portion of the process. Light propagatingfrom the diffuser enters the collimating lens 2009 and propagatesthrough the collimating lens 2009, and the light propagating from thecollimating lens 2009 is more collimated than the light received by thecollimating lens 2009. At block 2435, a microlens array receives lightfrom the collimating optical element, and light propagating from themicrolens array is provided towards a target to illuminate the target.In some embodiments, the target comprises a flow cell. The micro-lensarray can modify the distribution of light, such as to increaseuniformity. By way of example, various lenslets (e.g., each of aplurality of lenslets) of the micro-lens array 2011 (FIG. 20 ) cansample (or receive) the incident illumination profile of light that ispropagating from the diffuser. Each sample can be imaged onto the fieldof view (FOV) at the plane of a flow cell 2230 (e.g., FIG. 20 ; flowcell 1830 FIG. 18 ) with the focusing lens 2015. The samples from thevarious lenslets (e.g., each lenslet) of the micro-lens array can addtogether to generate a flat-top profile in the plane of the flow cell.An example of such an intensity profile is illustrated in FIG. 19B.

FIGS. 20 and 22 illustrate optical systems for generating a beam foranalyzing samples using laser-induced fluorescence. FIGS. 25-42 alsorelate to optical systems for generating a light beam with a shaped beamprofile that can be used for analyzing samples, where the beam incidenton the sample (e.g., the sample plane) is a flat top line beam. Theoptical systems illustrated in FIGS. 25, 29, 33, and 41 can include manyof the same components, and configurations, as the systems in FIGS. 20and 22 even if they are not specifically mentioned in the description ofFIGS. 25, 29, 33, and 41 , and thus the description will focus ondifferences in the systems for generating a flat top line beam.

FIG. 25 illustrates certain components and the incident light path of anembodiment of an optical system 2500 configured to generate a beam foranalyzing samples (e.g., biological and/or chemical samples) usinglaser-induced fluorescence, the optical system including a MEMS mirror2509 and a cylindrical lens array (CLA) 2513. Various implementations ofthe optical system 2500 may include more components or fewer components(for example, as illustrated in FIG. 43 ). As illustrated in the exampleof FIG. 25 , the system 2500 includes three laser diodes 2501A, 2501B,2501C which emit light that is initially in three separate opticalpaths. Although this example includes three lasers, other systems mayinclude two lasers, more than three lasers, or one laser. In an example,the lasers are multimode laser diodes. Light from each laser diode2501A, 2501B, 2501C propagates through a laser collimator 2503A, 2503B,2503C and through beam conditioning optics 2505A, 2505B, 2505C to adichroic beam combiner 2505 which combines the beams from the laserdiodes. In an example, the beam conditioning optics can 2504 can includeanamorphic prisms. The beam conditioning optics 2505 condition the lightbeam emitted by the laser to have a more elliptical shape. For example,changing the shape of the light beam (e.g., the beam profile) from agenerally circular shape to an elliptical shape. Changing the shape ofthe beam helps to fill the CLA with the incident beam. That is, the beamis shaped such that is spans across more of the lenslets of the CLA,which increases the uniformity and helps to better form the desired flattop line beam profile, of the light beam output from the CLA. In systemshaving one laser, a beam combiner is not needed. The combined beampropagates through a focusing lens 2507 to the MEMS mirror 2509, whichreflects the combined beam, based on a MEMS actuation process, to acollimating lens 2511. The beam propagates through the collimating lens2511 to the CLA 2513. The CLA 2513 can include a one-dimensional lensarray that includes an arrangement of cylindrical lenses that generate aflat top line beam from the light beam from the MEMS mirror 2509. TheCLA shapes the beam to produce a flat top line beam (for examples, thebeam profile illustrated in FIGS. 26, 27, and 28 ). The movement of theincident light beam on the plurality of lenses in the CLA helps tocreate uniformity in the irradiance of the flat top line beam. Each ofthe lenses creates a beam that is (at least partially) superimposed onthe other, producing a mixing effect the provides for uniformity. Thelight beam output from the CLA 2513, now a flat top line beam (e.g., asillustrated in FIGS. 26, 27, and 28 ) propagates through an objectivelens system 2515 (which may include one or more optical elements) and isincident on the sample plane 2517. The beam profile of the beam on thesample plane 2517 is a flat top line beam, where the flat top portion isuniform (or substantially uniform), as illustrated in FIG. 26 . In otherwords, the uniformity of the flat top portion of the flat top line beamis more uniform due to the movement of the beam by a movable mirrorsystem. The movable mirror system can be a MEMS mirror 2509 asillustrated in FIG. 25 , or a mirror galvanometer, or the like. For easeof reference, a movable mirror system is generally referred to herein asa MEMS mirror. The movement of the MEMS mirror can be, for example, at afrequency of between about 5 Hz to 50 kHz. In some examples, thefrequency is between 50 Hz and 10 kHz. In another example, the frequencyis between 100 Hz and 10 kHz. In another example, the frequency isbetween 500 Hz and 10 kHz. In some embodiments, the system 2500 may alsoinclude a diffuser optical element to add additional diffusion to thecombined beam. For example, system 2500 can include an engineereddiffuser 2512 positioned between the collimating lens 2511 and the CLA2513. The diffuser 2512 can be a 1D engineered diffuser. When theoptical system 2500 includes the engineered diffuser 2512, the beamconditioning optics need not include the anamorphic prisms in the beamconditioning optics, although some embodiments can include both theengineered diffuser and the anamorphic prisms. The 1D engineereddiffuser (and the anamorphic prisms) provide a formed diffuse light beamto the CLA to produce a more “filled” light beam on the CLA, that is, alight beam with more uniformity and less irregularity when measuredalong an axis. In some embodiments, the CLA includes a pair of 1Dcylindrical lens arrays spaced apart and having the axis of thecylindrical lenses aligned in the same direction. In some embodiments,the optical system does not include the focusing lens 2507 and/or thecollimating lens 2511 (see for example the optical system in FIG. 43 ).As illustrated in FIG. 33 , in some examples the optical system canincludes a lens 2523 (e.g., a cylindrical lens) positioned between theengineered diffuser 2512 and the CLA 2513

In these embodiments, the MEMS mirror 2509 is used instead of the movingdiffuser 2007 (e.g., FIG. 20 ) to diffuse the incident beam and increaseuniformity of the flat top line beam, for example, to reduce the effectsof speckle caused by the coherent light sources (i.e., lasers). In anyof the embodiments described herein, in some implementations a mirrorgalvanometer is used instead of the MEMS mirror. The MEMS mirror 2509can be configured to operate as a single-axis one-dimensional type.Although a single-axis movement of the light beam may generally bepreferred, in various embodiments, the MEMS mirror 2509 can be adual-axis two-dimensional type. In examples of dual-axis MEMS mirrorimplementations, the movement of the mirror in a one direction is muchless than in a second direction as the moving beam propagates to acylindrical array of lenses and the system is designed to form lightbeams with a flat top line beam profile having substantially uniformirradiance along the flat-top of the line beam (as uniform as possible).The MEMS mirror 2509 includes a movable reflective surface (mirror) thatis moved (e.g., rapidly) using current (e.g., for a single-axis MEMSmirror) or current and a magnetic field (e.g., for a dual-axis MEMSmirror). There are many types of MEMS mirror devices (which may bereferred to herein as a “MEMS mirror” for ease of reference) from avariety of manufacturers. A MEMS mirror typically operates using lowpower and can provide very rapid optical beam steering, in one-dimensionor two-dimensions. For example, various configurations of a MEMS mirrorcan include an actuatable mirror that deflect laser beams at deflectionangles of 0° up to plus/minus 5°, 10°, 15°, 25°, or 32°, at high speedsand along one or two-axes. In reference to the examples relating toFIGS. 25-42 , the MEMS mirror 2509 is positioned in the optical system2500 to receive a light beam from a focusing lens 2507. The focus of thelight beam received at the MEMS mirror 2509 from the focusing lens 2507does not need to be exactly at the surface of the MEMS mirror 2509,although it can be. Instead, the focus of the light beam from thefocusing lens 2507 can be before or after the MEMS mirror 2509 (e.g.,the MEMS mirror 2509 can be positioned at a distance, less than orgreater than, or at, the focal length of the focusing lens 2507, so longas the light beam on the MEMS mirror 2509 fits on the MEMS mirror 2509and does not overfill the MEMS mirror 2509. The MEMS mirror 2509reflects the beam from the focusing lens 2507 to a collimating lens2511. Some examples of such optical systems do not include a collimatinglens 2511. In operation, the MEMS mirror 2509 is actuated to move theincident beam such that the beam reflected from the MEMS mirror 2509moves a distance (e.g., back and forth, or in some other pattern) on thesurface of the CLA 2513 to provide a more diffuse light to the objectivelens 2515 and subsequently to the sample plane 2517. Reference to theMEMS mirror 2509 being at 45°, 45.5°, or 46° indicates the mirror is atinitial position of 45° relative to the incident light beam (e.g., basedon the positioning of the MEMS mirror housing), and then actuated tomove to be in a position of 45.5° or 46° relative to the incident lightbeam. These angles are merely examples, other angles can be used. Forexample, an angle for moving the MEMS mirror can be determined such thatthe angle corresponds with a desired movement distance of the light beamon the CLA 2513, e.g., movement of the centroid of the light beam acrossa certain portion of a lens of the CLA 2513.

In the example configuration of FIG. 25 , the MEMS mirror 2509 isinitially positioned at a 45° angle relative to incident light receivedat the MEMS mirror from the focusing lens. FIGS. 26-28 illustrate beamprofiles at the sample plane of this configuration (MEMS mirrorpositioned at a 45° angle). FIGS. 29-32 relate to a similar opticalsystem as illustrated in FIG. 25 , where the MEMS mirror is positionedat a 45.5° angle. FIGS. 33-36 relate to a similar optical system asillustrated in FIG. 25 , and the resulting beam profiles, where the MEMSmirror is positioned at a 46° angle. In this example, the MEMS mirror2509 is driven by signals provided by a controller 2510, which can be aseparate controller or incorporated into, or part of, another system(e.g., another computer system). The controller 2510 drives the MEMSmirror 2509 to move the mirror at a certain frequency, and a certainamount (e.g., in degrees) in one direction, or in two directions (e.g.,tip and tilt). The controller 2510 can drive the MEMS mirror 2509 basedon predetermined drive information that is stored on the controller 2510or provided to the controller. The drive information can include thefrequency of the mirror movement, the amount of mirror movement (+/−)along a first axis and/or along a second axis orthogonal to the firstaxis. In some embodiments, the predetermined information is generated bymeasuring one or more characteristics of the beam formed at the sampleplane (or at the objective lens, or another plane in the light pathafter the MEMS mirror) and adjusting the movement of the mirror toproduce the desired beam profile. For example, the flat top line beamcan be measured, and the movement of the mirror can be changed toincrease (or optimize) the uniformity of irradiance at the flat top ofthe line beam. In an example implementation, the flat top line beam canbe measured (e.g., that the sample plane) and the movement of the mirrorcan be changed automatically using a feedback loop between the measuringsensor and the controller to automatically change (e.g., increase oroptimize) the uniformity of irradiance at the flat top of the line beam.In some examples, various samples can be positioned at the sample plane,and the controller 2510 can be adjusted to produce a flat top line beamthat best reads the samples, and this information can be electronicallysaved as a particular beam profile for that type of sample.

FIG. 26 is a diagram illustrating an example of the X and Y-coordinatebeam profile at the sample plane of the optical system of FIG. 25 , theMEMS mirror being positioned at a 45° angle relative to incident lightreceived at the MEMS mirror from the focusing lens. Unlike the beamgenerated by the optical systems of FIGS. 20 and 22 , this beam has athin line profile which is useful for scanning certain types of samplessuch as in flow cells.

FIG. 27 is a graph illustrating an example of irradiance along thelong-axis beam profile (e.g., the Y coordinate value) at the sampleplane of the system of FIG. 25 when the MEMS mirror is positioned at a45° angle relative to incident light received at the MEMS mirror fromthe focusing lens. Along the Y coordinate, the irradiance curve shows asharp transition from near zero values to a flat-top portion havingsmall irregularities centered around the “0” Y coordinate value. FIG. 28is a graph illustrating an example of the irradiance along theshort-axis beam profile (e.g., the X coordinate value) at the sampleplane of the system of FIG. 25 . The irradiance along the X coordinateis more of a steep Gaussian curve centered at the “0” X coordinatevalue.

FIG. 29 illustrates certain components and the incident light path of anembodiment of the optical system of FIG. 25 , where the MEMS mirror ispositioned at a 45.5° angle relative to incident light received at theMEMS mirror from the focusing lens. FIG. 30 is a diagram illustrating anexample of the X and Y-coordinate beam profile at the sample plane ofthe optical system of FIG. 25 , the MEMS mirror being positioned at a45.5° angle relative to incident light received at the MEMS mirror fromthe focusing lens. FIG. 31 is a graph illustrating an example ofirradiance along the long-axis beam profile (e.g., the Y coordinatevalue) at the sample plane of the system of FIG. 29 when the MEMS mirror2509 is positioned at a 45.5° angle relative to incident light receivedat the MEMS mirror 2509 from the focusing lens. Along the Y coordinate,the irradiance curve again shows a sharp transition from near zerovalues to a flat-top portion having small and different irregularitiescentered around the “0” Y coordinate, the irregularities being differentthan those shown in FIG. 27 with the MEMS mirror 2509 at 45°.

FIG. 32 is a graph illustrating an example of irradiance along theshort-axis beam profile (e.g., the X coordinate value) at the sampleplane of the system of FIG. 29 , the MEMS mirror 2509 being positionedat a 45.5° angle relative to incident light received at the MEMS mirror2509 from the focusing lens. The irradiance along the X coordinate ismore of a steep Gaussian curve centered at the “0” X coordinate value.In various embodiments, the MEMS mirror can be moved to change the angleof incident of the light beam on the CLA 2513 in the range of plus orminus 0.001°, 0.002°, 0.003°, 0.004°, 0.005°, 0.006°, 0.007°, 0.008°,0.009°, or 0.010°. In some embodiments, the MEMS mirror can be moved tochange the angle of incident of the light beam on the CLA 2513 in therange of plus or minus 0.01°, 0.02°, 0.03°, 0.04°, 0.05°, 0.06°, 0.07°,0.08°, 0.09°, or 0.11°. In some embodiments, the MEMS mirror can bemoved to change the angle of incident of the light beam on the CLA 2513in the range of plus or minus 0.1°, 0.2°, 0.3°, 0.4°, 0.5°, 0.6°, 0.7°,0.8°, 0.9°, or 1.0°. In some embodiments, the MEMS mirror 2509 can bemoved to change the angle of incident of the light beam on the CLA 2513in the range of plus or minus 1°, 2°, 3°, 4°, 5°, 6°, 7°, 8°, 9°, or10°, or greater than 10°. The angle determines how much the beam moveson the lens array. In various embodiments, the MEMS mirror 2509 can movethe light beam to any angle that is just less than the acceptance angleof the CLA 2513.

FIG. 33 illustrates certain components and the incident light path of anembodiment of the optical system of FIG. 25 , where the MEMS mirror ispositioned at a 46° angle relative to incident light received at theMEMS mirror from the focusing lens. FIG. 33 also illustrates that theoptical system may include a lens 2523 positioned between an engineereddiffuser 2512 and the CLA 2513. The lens 2523 can be a cylindrical lenswhich focuses light from the diffuser into a line at the CLA 2513. FIG.34 is a diagram illustrating an example of the X and Y-coordinate beamprofile at the sample plane of the optical system of FIG. 33 , the MEMSmirror being positioned at a 46° angle relative to incident lightreceived at the MEMS mirror from the focusing lens. FIG. 35 is a graphillustrating an example of irradiance along the long-axis beam profile(e.g., the Y coordinate value) at the sample plane of the system of FIG.33 when the MEMS mirror is positioned at a 46° angle relative toincident light received at the MEMS mirror from the focusing lens. Alongthe Y coordinate, the irradiance curve again shows a sharp transitionfrom near zero values to a flat-top portion having small and differentirregularities centered around the “0” Y coordinate, the irregularitiesbeing different than those shown in FIGS. 27 and 31 with the MEMS mirrorat 45° and 45.5°, respectively. The irradiance profile in FIG. 35 showssome sag in the profile (e.g., from left-to-right in the graph), whichmay be due to clipping in the objective lens. FIG. 36 is a graphillustrating an example of the irradiance along the short-axis beamprofile (e.g., the X coordinate value) at the sample plane of the systemof FIG. 33 . Again, irradiance along the X coordinate is a steepGaussian curve centered at the “0” X coordinate value.

FIG. 37 is a graph illustrating the Y-Centroid position of the beam (mm)on the CLA (y-axis) as a function of the MEMS mirror tilt (x-axis),showing this to be a linear relationship. In an example, the CLA 2513includes a 1D array of cylindrical lenses. Actuating the MEMS mirror2509 moves the centroid of the beam to be at different positions on thesurface of the CLA. Depending on the implementation, the movement of thebeam may move the centroid across a portion of a lens of the CLA, oracross a whole lens. In some embodiments, the fraction of a lens whichthe beam is displaced across may be determined empirically. The lensesof the CLA can be configured to have a certain pitch, array size length,array size width, and array size thickness. Each lens in the CLA isconfigured to have a pitch, focal length, and diameter. In someembodiments, the Y centroid position of the beam on the CLA is moved bythe MEMS mirror 2509 across about 1% or less of a dimension of a lens inthe CLA (e.g., a diameter of a lens). In other embodiments, the Ycentroid position of the beam on the CLA is moved by the MEMS mirror2509 across about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%,13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%,27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%,41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%,69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99%, or 100%, (plus or minus 0.05%) of a lens in the CLA. Insome embodiments, the Y centroid position of the beam on the CLA ismoved more than one lens.

FIG. 38 is a graph illustrating the cylindrical lens array (CLA) Y-AoI(y-axis) as a function of the MEMS mirror tilt (x-axis). This graphshows that the angle of incidence of the beam on the CLA (reflected fromthe MEMS mirror) changes only slightly with MEMS mirror tilt, whichprevents (or at least partially prevents) side lobes from appearing.Side lobes appear if the incident beam angle on the CLA is greater thanthe CLA acceptance angle. In this example, the Y-AoI changes linearlyfrom about 0.000 degrees to about 0.002 degrees as the mirror tiltchanges from 0.0 degrees to 1 degree.

FIG. 39 is a graph illustrating the Y-Centroid position (mm) of the beamon the objective lens (y-axis) as a function of the MEMS mirror tilt(x-axis). In this example, the Y-centroid position changes linearly withrespect to the mirror tilt, from an initial Y-centroid position of 0.0to about −1.05 mm as the mirror is tilted from 0 degrees to 1.0 degree.At some point the beam will start to clip on the objective lens stop,which will result in line uniformity degradation.

FIG. 40 is a graph illustrating the objective lens Y-AoI (y-axis) as afunction of the MEMS mirror tilt (x-axis). This graph shows thatobjective Y-AoI changes only slightly with MEMS mirror tilt, which isimportant for maintaining the position of the illumination field-of-view(FOV) as the MEMS mirror moves. As can be seen in this graph, theobjective AoI changes only slightly with MEMS mirror tilt, which iscritical for maintaining the position of the illumination FOV as theMEMS mirror moves.

FIG. 41 illustrates the incident light path of an embodiment of anoptical system 4100 generally similar to the optical system of FIG. 25 ,including having a MEMS mirror 2509 in the beam path between a focusinglens 2507 and a collimating lens 2511. System 4100 also is configured togenerate a flat top line beam. However, in system 4100 the CLA has beenreplaced by a diffuser 2514 positioned at a distance d₁ from thecollimating lens and at a distance d₂ from the objective lens. In someembodiments, the diffuser 2514 is an engineered diffuser. In someembodiments, the use of an engineered diffuser allows the elimination ofprisms in the beam conditioning optics. For example, a 1D diffuser canbe used to “fill” the lens array (i.e., provide light across a portionof the CLA, or across all of the CLA). In some embodiments, acylindrical lens can be used to fill the CLA. The engineered diffusercan be configured to diffuse light in one direction but not in anorthogonal direction. For example, to diffuse light in the Y-directionbut not diffuse light in the X-direction (or provide a much lowerdiffusion of light in the X-direction). As illustrated in FIG. 41 , insome embodiments, an engineered diffuser can provide sufficientdiffusing and beam shaping to and is used in place of the CLA (see forexample, FIG. 41 ). In this case, the movement of the light beam by theMEMS mirror provides dithering to smooth out the high spatial frequencynoise we see on the output of the engineered diffuser. In otherembodiments, an engineered diffuser can be positioned in the light pathbefore the CLA and is used to fill the linear cylindrical lens arraywithout affecting the orthogonal axis.

FIG. 42 is a schematic that illustrates an example of a cylindrical lensarray 2513 (CLA). In an example, the CLA has a single element 2513 athat is a one-dimensional (1D) array having cylindrical lenses. Inanother example, the CLA has a pair of cylindrical lens arrays, CLAelement 2513 a and CLA element 2513 b, that are spaced apart, eachelement being a one-dimensional (1D) array having cylindrical lenses.With such CLAs, the shaping of the light is on one axis such that theoutput beam is a flat top line beam. In the example shown in FIG. 42 ,each of the cylindrical lenses of the CLA has a diameter distance d. Asindicated above, the MEMS mirror 2509 can be driven to move theY-centroid position of the beam on the CLA a certain distance, thedistance being less than or equal to the diameter distance d.

FIG. 43 illustrates an embodiment of an optical system 2550 configuredto generate a flat top line beam for analyzing samples the opticalsystem including a MEMS mirror and a cylindrical lens array (CLA). Thissystem includes many of the elements of the optical system illustratedin FIG. 25 , for example, one or more lasers 2501, and for each laser acollimator 2503, beam conditioning optics 2505, and a beam combiner2505. The optical system 2550 also includes a mirror system (MEMS mirror2509) and a CLA 2513, and an objective lens 2515. Unlike the opticalsystem of FIG. 25 , the example of an optical system in FIG. 43 does notinclude a collimating lens between the MEMS mirror and the CLA, and doesnot include a focusing lens between the beam combiner and the MEMSmirror. In some embodiments, the beam conditioning optics 2505 canincludes anamorphic prisms. In some embodiments, the beam conditioningoptics 2505 includes an engineered diffuser instead of the prisms. Whenthe beam conditioning optics includes an engineered diffuser, dependingon the implementation the optical system may not include a focusing lens2507 or a collimating lens 2511, or it may include one or both of thefocusing lens 2507 and the collimating lens 2511.

In various embodiments, including any of the embodiments described aboveor elsewhere herein, an optical fiber can be included in this system totransport light from one location to another. Accordingly, an opticalfiber may be included, for example, in an optical path between any twoelements instead of propagating light in free space. Accordingly, anoptical fiber may be included at different locations within the opticalsystem. Additionally, such optical fiber may be included in one or morelocations within the optical system. Likewise, one or more opticalfibers may be included in the optical system.

An optical fiber, for example, can be positioned in the optical pathbetween the light source (e.g., laser diodes 2501A, 2501B, and 2501C)and one or more downstream optical component such that the light sourcecan be remote from the sample plane 2517 and/or the sample. In variousimplementations, for example, the laser source (e.g., laser diodes2501A, 2501B, and 2501C) can be located a distance of between 10centimeters (cm) and 5 meters or 50 centimeters and 1 meter or 1 meterand 5 meters from the sample plane 2417 and/or the sample beingilluminated with laser light and/or evaluated or any range of distancesformed by any of these values or the distance may be outside theseranges in some implementations. As shown in FIG. 44 , for example, anoptical fiber 2520 is disposed in an optical path between the laserdiodes 2501A, 2501B, and 2501C and the moveable mirror system or MEMSmirror 2509. In particular, in this example, the optical fiber 2520 isdisposed in an optical path between one of the dichroic beam combiners2505 and a lens such as a focusing lens 2507 disposed between theoptical fiber and the MEMS mirror 2509. The focusing lens 2507 maycomprise, for example, a positive power lens having a positive focallength and may be disposed with respect to the optical fiber 2520 andthe MEMS mirror 2509 to received light from the optical fiber and focuslight onto the MEMS mirror. In various implementations, for example, theoptical fiber 2520 has a first end disposed to receive light from thelight sources and a second end disposed to output light to the MEMSmirror 2509.

The optical fiber 2520 may comprise a core and a cladding and may be asingle or multimode optical fiber. The optical fiber 2520 may have acore of any shape, including circular, square or rectangular. A fiberoptic 2520 with a square or rectangular core may advantageously provideincreased homogenization of the laser light prior to being incident onthe MEMS mirror 2509 within the optical imaging system and potentiallyyield a more uniform flat-top line profile. Corners of the square orrectangle where sides of the core meet may, for example, provide forincreased homogenization. In various implementations, the cross-sectionof the core orthogonal to its length can have a width, diameter, height,or any combination of these, that is between 5 μm and 600 μm for oneaxis and 5 μm and 600 μm for the orthogonal axis. For example, a 150μm×450 μm fiber could be used with the laser beams scanning via the MEMSmirror along the 450 μm axis. The optical fiber 2520 may have flexibleand/or easily bendable as opposed to being rigid and unable to be benteasily. The material of the optic fiber 2520 may comprise plastic, glassor fused silica or other material(s).

As shown in FIG. 44 , light from the laser diodes 2501A, 2501B, and2501C propagates through respective laser collimators 2503A, 2503B,2503C and beam conditioning optics 2505A, 2505B, 2505C to dichroic beamcombiners 2505A, 2505B, which combine light from the different lightsources (e.g., laser diodes). In systems having one laser, a beamcombiner is not needed. The light from the light sources 2501A, 2501B,and 2501C is input into the first (e.g., proximal) end of the fiberoptic 2520 and exits through the second (e.g., distal) end of theoptical fiber. The light output from the second end of the optical fiber2520 is directed onto the MEMS mirror 2509, which reflects the light tothe cylindrical lens array 2513. In the design shown in FIG. 44 , thefocusing lens 2507 causes light output from the second end of theoptical fiber 2520 to converge prior to being incident on the MEMSmirror 2509. Accordingly, in various implementations, the focusing lens2507 focuses light exiting the optical fiber 2520 onto the MEMS mirror2509.

In another example, as shown in FIG. 45 , for example, an optical fiber2520 is disposed in an optical path between the MEMS mirror 2509 and themicrolens array 2513. In various implementations, the optical fiber 2520has a first end disposed to receive light from the MEMS mirror 2509 anda second end disposed to output light to the microlens array 2513. Inparticular, in this example, a lens such as a focusing lens 2505 isdisposed in an optical path between the MEMS mirror 2509 and the opticalfiber 2520. The focusing lens 2507 may comprise for example a positivepower lens having a positive focal length and may be disposed withrespect to the optical fiber 2520 and the MEMS mirror 2509 to receivedlight from the MEMS mirror and focus light onto the optical fiber 2520.In some implementations, the lens may be positioned a focal length awayfrom the first (input) end of the optical fiber 2520. In variousimplementation, the light from the light sources that is incident on andreflected from the MEMS mirror 2509 is collimated. Such collimated lightmay be incident on the focusing lens 4505 and be focused down, forexample, onto the first/input end of the optical fiber 2520. Anotherlens, such as collection lens and/or collimating lens 2511 may receivelight exiting the second (output) end of the optical fiber 2520. Thiscollimating lens 2511 may also comprise a positive power lens having apositive focal length, in some embodiments. In some configurations, thiscollimating lens 2511 is position a distance from the second/exit end ofthe optical fiber that corresponds to the focal length of thecollimating lens. Accordingly, the light from the optical fiber 2520collected by the collimating lens 2511 may having increased collimationwhen incident on the microlens array.

The optical fiber 2520 may comprise a core and a cladding and may be asingle or multimode optical fiber in various implementations. Theoptical fiber 2520 may have a core of any shape, including circular,square or rectangular. A fiber optic 2520 with a square or rectangularcore may advantageously provide increased homogenization of the laserlight prior to being incident on the MEMS mirror 2509 within the opticalimaging system and may potentially yield a more uniform flat-top lineprofile. Corners of the square or rectangle, where sides of the coremeet, may for example provide for increased homogenization. In variousimplementations, the cross-section of the core orthogonal to its lengthcan have a width, diameter, height, or any combination of these, that isbetween 5 μm and 600 μm for one axis and 5 μm and 600 μm for theorthogonal axis. For example, a 150 μm×450 μm fiber could be used withthe laser beams scanning via the MEMS mirror along the 450 μm axis. Invarious implementations, the optical fiber 2520 may be flexible and/oreasily bendable as opposed to being rigid and unable to be bent easily.In some implementations, the material of the optic fiber 2520 maycomprise plastic, glass or fused silica or other material(s).

In various implementations, the optical fiber can provide forappropriate entendue. For example, in various implementations, e.g.,where the MEMS mirror is upstream of the fiber, to achieve a 50 μm×1 mmline profile at the flow cell if a 0.7 numerical aperture (NA) objectivelens is being used with a 0.22 NA fiber, a fiber having a core with awidth along the small axis that is no larger than ˜(0.7/0.22) 50 μm=159μm can be employed in some cases. This relationship applies to the smallaxis of the line in this example. The larger axis can be larger sizethan the size set forth in this relationship. Accordingly, in variousimplementations, the size of the core, e.g., the width of the core suchas in the direction or along the axis of the core that has a smallerwidth/height or lateral dimension (e.g., x direction), may be less thanor equal to the product of the width of the output beam at the sample orsample plane along the shortest direction and the ratio of the numericalaperture of the objective lens to the numerical aperture of the opticalfiber (i.e., the numerical aperture of the objective lens divided by thenumerical aperture of the optical fiber). For example, the width of thecore such as in the direction or along the axis of the core that issmallest may be less than or equal to(NA_(objective)/NA_(fiber))×width_(beam), where NA_(objective) is thenumerical aperture of the objective lens, NA_(fiber) is the numericalaperture of the fiber, and width_(beam) is the width of the beam in thedirection of the shortest width of the beam cross-section at the sampleor sample plane. Other designs are possible.

As discussed above, the MEMS mirror 2509 can be scanned, for examplealong one direction (such as the y-direction) and/or may be scanned thefastest along this direction and/or the most (e.g., largest distance orover largest angle) in this direction. In some implementations, this onedirection is the only direction that the MEMS mirror tilts or scans. Invarious implementations, the incident beam on the MEMs mirror 2509 isscanned in one direction (possibly more) through the focusing lens 4505,such that the beam moves laterally across the first/input end of theoptical fiber 2520, e.g., across the input face of the optical fiber.The position on the core of the optical fiber 2520 at which the light isincident and input into the core may thus be scanned and change withtime and thereby may excite different optical modes of the fiber overtime. Such designs may therefore advantageously provide a time-varyingpattern on the output end of the optical fiber 2520 that forms ahomogenous light distribution (at the second/output end of the fiber aswell as at the sample plane and sample) when measured or integrated overa time period longer than the period of the scanning of the MEMs mirror2509. This light output from the second/output end of the optical fiber2520 may then propagate to the micro-lens array 2513. In theimplementation shown in FIG. 45 , this light passes through thecollimating lens 2511 onto the micro-lens array 2513 increasing thecollimation of the light incident on the micro-lens array.

In the example shown in FIG. 45 , the microlens array comprises acylindrical lens array having optical power along the long direction ofthe beam spot at the sample plane (e.g., in the y-direction) and zerooptical power along the short direction of the beam spot at the sampleplane (e.g., in the x direction). In some implementations, this longdirection may also correspond to the direction the MEMS mirror is tiltedor tilted fastest and/or most. In some implementations, this longdirection is the only direction that the MEMS mirror tilts or scans.Accordingly, in various implementations such as shown in FIG. 45 , themicrolens array primarily or only affects the beam along the MEMS scandirection or along the axis coinciding with the MEMS scan direction(e.g., y direction). In the example shown in FIG. 45 , this directioncorresponds to the longer direction of the cross-section of the core ofthe fiber (e.g., the direction that is 450 μm in width, for the example,for a core that has rectangular cross-section having a height and widthof 150 μm×450 μm). The microlens array does not affect or does notaffect as much the beam or beam shape in the shorter direction of thecross-section of the core (e.g., the direction that is 150 μm in heightfor the example core that is 150 μm×450 μm). The light may, for example,be focused in the y direction but not in the x direction by themicrolens array and travels to a focusing lens and an image is formed atthe sample plane. With the combination of lenses (e.g., the collimatinglens 2511 and the focusing lens 2515) shown in FIG. 45 , the size of thebeam in the shorter direction is reduced, e.g., by a demagnification of⅓ to 50 μm. The microlens array angle, in conjunction with the focusinglens 2515, can be chosen to generate the desired length of the line inthe long direction (y-direction) at the sample plane. In this example,this length of the spot at the sample plane is about 1 mm along the longaxis (y direction). As a result, in this example, a flat-top line ofabout 1 mm×50 um is formed.

In any of the examples above or elsewhere herein, one or more of thelenses such as focusing lenses, collimating lenses, etc. may includecolor correction to reduce chromatic aberration. Accordingly, suchlenses may comprise achromats such as achromatic doublets in variousimplementations. In some implementations, the micro-lens array maycomprise one or more cylindrical lens arrays.

FIG. 46 shows a design that includes a single achromat (e.g., anachromatic doublet) 2511 collimating light exiting the second/output endof the optical fiber 2520. As discussed above, in variousimplementations, this collimating lens 2511 comprises a positive powerlens with a positive focal length. In some implementations, the lens ispositioned a distance from the second/output end of the optical fiber2520 corresponding to the focal length. In the design shown, forexample, the collimating lens has a focal length of 35 mm. Thecollimating lens 2511 may in some implementations be rotationallysymmetrical and may have the same optical power in orthogonaldirections, such as x and y directions. Accordingly, in variousimplementations, this collimating lens 2511 is not a cylindrical lens oran anamorphic lens. In various implementations, this collimating lens2511 is an achromatic such as an achromatic doublet.

FIG. 47 shows a close-up view of the system of FIG. 46 depicting MEMSmirror 2509 directing light through the focusing lens 4505 onto theinput face of the optical fiber 2520. The changing orientation of theMEMS mirror 2509 thereby positions the light beam input into the core ofthe optical fiber 2520 at various positions across the core of the fiberat various times as the MEMS mirror is scanned. The focusing lens 4505in this example comprises an achromat (e.g., an achromatic doublet)having an effective focal length of 4.5 mm.

FIG. 48 is a diagram illustrating an example of the X and Y-coordinatebeam profile at the sample plane of the optical system of FIGS. 46 and47 . This beam has a thin line profile which is useful for scanningcertain types of samples such as in flow cells.

FIG. 49 is a graph illustrating an example of the irradiance along theshort-axis beam profile (e.g., the X coordinate value) at the sampleplane of the system of FIG. 48 .

FIG. 50 is a graph illustrating an example of irradiance along thelong-axis beam profile (e.g., the Y coordinate value) at the sampleplane of the system of FIG. 48 . Along the Y coordinate, the irradiancecurve shows a sharp transition from near zero values to an approximatelyflat-top portion having somewhat higher values around the “0” Ycoordinate value.

FIG. 51 shows a design that includes a first cylindrical collimatinglens 4510A and a second cylindrical collimating lens 4510B collimatinglight exiting the second/output end of the optical fiber 2520. Invarious implementations, the first cylindrical collimating lens 4510Acomprises optical power in a first direction (e.g., y-direction) and notin the orthogonal second direction (e.g., x-direction), while the secondcylindrical collimating lens 4510B comprise optical power in the secondorthogonal direction (e.g., x-direction) and not in the first direction(e.g., y-direction) at the sample plane (e.g., at the flow cell). In theexample design shown, the first cylindrical collimating lens 4510A hasan effective focal length of 10 mm along the direction where the beam atthe sample plane is widest and/or in the direction of the MEMS mirrorscan and/or in the direction where the MEMS mirror scans the fastestand/or the widest or most distance (e.g., along the y-direction) and thesecond cylindrical collimating lens 4510B has an effective focal lengthof 30 mm the orthogonal axis (y-axis) at the sample plan (e.g., at theflow cell). In some implementations, the effective focal length of thesecond cylindrical collimating lens 4510B is 35 mm. Other effectivefocal lengths, however, may be used.

As discussed above, in various implementation this collimating lenscomprises a positive power lens with a positive focal length. In someimplementations, the lens is positioned a distance from thesecond/output end of the optical fiber 2520 corresponding to the focallength.

FIG. 52 is a diagram illustrating an example of the X and Y-coordinatebeam profile at the sample plane of the optical system of FIG. 51 . Thisbeam has a thin line profile which is useful for scanning certain typesof samples such as in flow cells.

FIG. 53 is a graph illustrating an example of the irradiance along theshort-axis beam profile (e.g., the X coordinate value) at the sampleplane of the system of FIG. 48 .

FIG. 54 is a graph illustrating an example of irradiance along thelong-axis beam profile (e.g., the Y coordinate value) at the sampleplane of the system of FIG. 48 . Along the Y coordinate, the irradiancecurve shows a sharp transition from near zero values to an approximatelyflat-top portion having some variation across.

Multi-Line Laser Systems

Various aspects of this disclosure relate generally to optical systemsfor directing light, such as to a sample contained in a flow cell.Various implementations can include one or more beam-splitting opticalelements, such as a prism assembly, which can separate a beam of lightinto multiple beams of light of different wavelengths. The embodimentsdisclosed in this section can use various features and componentsdisclosed in connection with other embodiments described herein, ordisclosed in the '348 application and/or the '213 patent. Variousfeatures and embodiments disclosed in this section can be incorporatedinto the systems and embodiments disclosed in connection with otherembodiments described herein, or disclosed in the '348 applicationand/or the '213 patent. By way of example, some embodiments can utilizethe lasers 2501A, 2501B, 2501C, the beam conditioning optics 2505, theoptical fiber 2520, the movable mirror (e.g., MEMS mirror 2509), themicrolens array 2513, the objective lens system 2515, and the sampleplane 2517, as disclosed in FIG. 45 of the '348 application, or in otherconfigurations disclosed therein. Additional components disclosed in the'348 application and/or the '213 patent can be used with the featuresand embodiments disclosed herein, such as collimating and/or focusinglenses, diffusers, vibrating or moving diffusers, controllers, Powelllenses, etc. Some embodiments disclosed herein show examples ofcomponents of the illumination system after the light exits an opticalfiber, which can be the optical fiber 2520.

In some embodiments, the illumination system can be configured toreceive light that is a combination of multiple wavelengths, and thesystem can be configured to split the light of different wavelengthsinto separate beams of light. The system can modify the shape and/ordistribution of light of the beams to produce output lines of differentwavelengths at different locations. The system can produce lines havinga substantially flat-top profile or distribution of light, similar tothe disclosure of the '348 application, except that light of differentwavelengths can be split into different, offset lines.

FIG. 55 is a side view of an example embodiment of an illuminationsystem 5000. FIG. 56 is a top view of the example embodiment of theillumination system 5000. FIGS. 55 and 56 show components of the system5000 after the light exits an optical fiber (not shown), although lightcan be provided by or to the system 5000 using various other components,as disclosed in the '348 application and/or the '213 patent. The lightoutput from the optical fiber can include light of a first wavelength(e.g., 525 nm or Green light) 5021 g, and also light of a secondwavelength (e.g., 635 nm or Red light) 5021 r. A first laser (not shown)can provide a laser beam of the first wavelength, which can be coupledinto the optical fiber, and a second laser (not shown) can provide alaser beam of the second wavelength, which can be coupled into theoptical fiber. The light output from the optical fiber can be divergingand/or can include the first wavelength of light mixed together with thesecond wavelength of light.

The system 5000 can have a collimating optical element 5011 (e.g., alens), which can be configured to at least partially collimate the lightoutput from the optical fiber. The light output from the collimatingoptical element 5011 can be more collimated than the light output fromthe optical fiber and/or received by the collimating optical element5011. The collimating optical element 5011 can output a combined beam oflight, which can include light of the first wavelength and light of thesecond wavelength (e.g., mixed together). The combined beam of light canbe considered to be a first beam of light of the first wavelength and asecond beam of light of the second wavelength, and the first and secondbeams of light can be on top of each other, coaxial, at least partiallyoverlapping, substantially completely overlapping, substantiallyparallel, etc. The combined beam of light can be provided by otherconfigurations, such as without an optical fiber (e.g., using multiplelasers and a beam combiner). In some embodiments, the collimatingoptical element 5011 can be omitted, such as if the light is alreadysufficiently collimated.

The system 5000 can include a prism assembly 5025, which can receive thecombined beam of light (e.g., from the collimating optical element5011). The prism assembly 5025 can be configured to split the light ofthe second wavelength apart from the light of the first wavelength. Theprism assembly 5025 can include multiple prisms, which can be coupledtogether to provide polychroic (e.g., dichroic) interfaces that can beconfigured to reflect the first wavelength of light and transmit thesecond wavelength of light, or vice versa. In some embodiments,polychroic coatings or other filters can be used (e.g., between theprism elements of the prism assembly 5025). The prism assembly 5025 candirect the different wavelengths of light along different optical pathsthrough the prism assembly, and can output the different wavelengths oflight at different locations and/or at different angles.

FIG. 57 is a detailed side or cross-sectional view of the prism assembly5025. The prism assembly 5025 can include a plurality of prism elements5026. In the example of FIG. 57 , the prism assembly 5025 includes fourprism elements 5026 a-d, although other numbers of prism elements 5026can be used, as discussed herein. A first prism 5026 a can be atriangular prism, having a first side A, a second side B, and a thirdside C. The sides of the prisms are indicated by letters with linespointing to the inside surfaces of the sides. A second prism 5026 b canbe a 4-sided or rhomboid prism, having a first side D, a second side E,a third side F, and a fourth side G. A third prism 5026 c can be afour-sided or rhomboid prism, having a first side H, a second side I, athird side J, and a fourth side K. A fourth prism 5026 d can be atriangular prism, having a first side L, a second side M, and a thirdside N. The prism assembly 5025 can have an aggregate cross-sectionalshape of a convex hexagon, such as an elongated hexagon, although otherconfigurations are possible.

The first side A of the first prism 5026 a can provide an input surfacefor receiving the combined beam of light into the prism assembly 5025(e.g., into the first prism 5026 a). The input surface can have ananti-reflection coating, in some implementations. The second side B ofthe first prism 5026 a can be coupled to the first side D of the secondprism 5026 b to form a first interface 5028. The first interface 5028can transmit light of the first wavelength (e.g., Green) 5021 g andreflect light of the second wavelength (e.g., Red) 5021 r. The firstinterface 5028 can include a polychroic (e.g., dichroic) filter, such asa coating applied to the outside of surface B or surface D. Thereflected light of the second wavelength can be directed toward thethird side C of the first prism 5026 a. The second side E of the secondprism 5026 b can be configured to reflect the first wavelength of light5021 g (e.g., towards the third side F of the second prism 5026 b). Insome embodiments, the second side E can reflect the light 5021 g bytotal internal reflection (e.g., due to air or another relatively lowindex material outside of side E), although in some embodiments areflective coating can be applied to side E. The third side C of thefirst prism 5026 a can be coupled to the first side H of the third prism5026 c to form a second interface 5030. The second interface 5030 cantransmit the second wavelength of light 5021 r from the first prism 5026a to the third prism 5026 c. In some embodiments, an index matchingmaterial can join side C and side H. The second surface I of the thirdprism 5026 c can reflect the second wavelength of light 5021 r (e.g.,toward the third side J of the third prism 5026 c). In some embodiments,the second side I can reflect the light 5021 r by total internalreflection (e.g., due to air or another relatively low index materialoutside of side I), although in some embodiments a reflective coatingcan be applied to side I. The first side L of the fourth prism 5026 dcan be coupled to the third side F of the second prism 5026 b, forming athird interface 5032. The third interface 5032 can transmit the firstwavelength of light 5021 g from the second prism 5026 b to the fourthprism 5026 d. In some embodiments, an index matching material can joinside F to side L. The third side J of the third prism 5026 c can becoupled to the second side M of the fourth prism 5026 d, forming afourth interface 5034. The fourth interface 5034 can reflect light ofthe first wavelength (e.g., Green) 5021 g and can transmit light of thesecond wavelength (e.g., Red) 5021 r. The fourth interface 5034 caninclude a polychroic (e.g., dichroic) filter, such as a coating appliedto the outside of surface J or surface M. The reflected light of thefirst wavelength can be directed toward the third side N of the fourthprism 5026 d. The side N can be an output surface for outputting thelight from the prism assembly 5025. The output surface can have ananti-reflection coating, in some implementations.

The prism assembly 5025 can output a first beam of light of the firstwavelength 5021 g and a second beam of light of the second wavelength5021 r. The first beam and the second beam output by the prism assembly5025 can be offset from each other. In some embodiments, the first beamand the second beam can partially overlap each other. In FIG. 55 , thecenter or axis of the first (e.g., Green) beam of light 5021 g exits theprism assembly 5025 at a first location, and the center or axis of thesecond (e.g., Red) beam of light 5021 r exits the prism assembly 5025 ata second location that is offset from (e.g., higher than) the firstlocation.

The first beam and the second beam output from the prism assembly 5025can be angled relative to each other and/or relative to the combinedbeam of light received by the prism assembly 5025. FIG. 58 shows anexample of the first light beam 5036 of the first wavelength of light(e.g., Green) 5021 g and the second light beam 5038 of the secondwavelength of light (e.g., Red) 5021 r being output from the prismelement 5026 d. In FIG. 58 , axis line 5040 is a line parallel to thedirection of the combined beam of light (e.g., that is input into theprism assembly). An angle 5042 between the first light beam 5036 and thesecond light beam 5038 can be about 0.5 milliradians (mrad), about 0.75mrad, about 1 mrad, about 1.5 mrad, about 2 mrad, about 3 mrad, about 5mrad, about 7 mrad, about 10 mrad, about 15 mrad, about 20 mrad, about25 mrad, about 30 mrad, about 35 mrad, about 40 mrad, about 45 mrad,about 50 mrad, or any values or ranges between any of these values(e.g., between about 1 mrad and about 30 mrad), although otherconfigurations are possible. The first beam 5036 and the second beam5038 can converge towards each other as they exit the prism assembly5025. One or both of the beams 5036 and 5038 can be angled relative tothe input combined beam of light. An angle 5044 between the first beam5036 and a line or axis 5040 parallel to the direction of the combinedbeam of light can be about 0.25 mrad, about 0.5 mrad, about 0.75 mrad,about 1 mrad, about 1.5 mrad, about 2 mrad, about 3 mrad, about 5 mrad,about 7 mrad, about 10 mrad, about 15 mrad, about 20 mrad, about 25mrad, about 30 mrad, or any values or ranges between any of thesevalues, although other configurations are possible. An angle 5046between the second beam 5038 and a line or axis 5040 parallel to thedirection of the combined beam of light can be about 0.25 mrad, about0.5 mrad, about 0.75 mrad, about 1 mrad, about 1.5 mrad, about 2 mrad,about 3 mrad, about 5 mrad, about 7 mrad, about 10 mrad, about 15 mrad,about 20 mrad, about 25 mrad, about 30 mrad, or any values or rangesbetween any of these values, although other configurations are possible.

The first beam 5036 and the second beam 5038 can cross so that the beamthat exits higher from the prism assembly 5025 produces the lower outputline, and so that the beam that exits lower from the prism assembly 5025produces the higher output line, as shown in FIG. 59 . The convergingbeams 5036 and 5038 can cross at an aperture or stop of the objectivelens system 5015 (e.g., for telecentricity), as discussed herein.

The angle of the first light beam 5036 can be produced by the reflectionat interface 5028 and/or by the reflection at surface I. If bothreflections were at 45 degrees, then the first light beam 5036 would beparallel with the combined input beam of light. In some embodiments, theinterface 5028 can be angled relative to the combined input beam by 45degrees plus or minus an offset amount of about 0.02 degrees, about 0.05degrees, about 0.1 degrees, about 0.2 degrees, about 0.3 degrees, about0.5 degrees, about 0.75 degrees, about 1 degree, about 1.25 degrees,about 1.5 degrees, about 1.75 degrees, about 2 degrees, about 2.5degrees, about 3 degrees, or any values or ranges there between (e.g.,between about 0.1 degrees and about 1 degree). With about 1 degree ofoffset, the interface 5028 can be angled at about 44 degrees or at about46 degrees, depending on the direction of the offset. The offset angleof the interface 5028 can angle the first output beam 5036 even if thesurface I is angled at 45 degrees relative to the combined input beam.In some embodiments, the surface I can angled relative to the combinedinput beam by 45 degrees plus or minus an offset amount of about 0.02degrees, about 0.05 degrees, about 0.1 degrees, about 0.2 degrees, about0.3 degrees, about 0.5 degrees, about 0.75 degrees, about 1 degree,about 1.25 degrees, about 1.5 degrees, about 1.75 degrees, about 2degrees, about 2.5 degrees, about 3 degrees, or any values or rangesthere between (e.g., between about 0.1 degrees and about 1 degree). Withabout 1 degree of offset, the surface I can be angled at about 44degrees or at about 46 degrees, depending on the direction of theoffset. The offset angle of the surface I can angle the first outputbeam 5036 even if the interface 5028 is angled at 45 degrees relative tothe combined input beam. In some embodiments, both the interface 5028and the surface I can be offset from 45 degrees. In some embodiments,the surface I can be angled relative to the interface 5028 by an angleof about 0.02 degrees, about 0.05 degrees, about 0.1 degrees, about 0.2degrees, about 0.3 degrees, about 0.5 degrees, about 0.75 degrees, about1 degree, about 1.25 degrees, about 1.5 degrees, about 1.75 degrees,about 2 degrees, about 2.5 degrees, about 3 degrees, or any values orranges there between (e.g., between about 0.1 degrees and about 1degree).

The angle of the second light beam 5038 can be produced by thereflection at interface 5034 and/or by the reflection at surface E. Ifboth reflections were at 45 degrees, then the second light beam 5038would be parallel with the combined input beam of light. In someembodiments, the interface 5034 can angled relative to the combinedinput beam by 45 degrees plus or minus an offset amount of about 0.02degrees, about 0.05 degrees, about 0.1 degrees, about 0.2 degrees, about0.3 degrees, about 0.5 degrees, about 0.75 degrees, about 1 degree,about 1.25 degrees, about 1.5 degrees, about 1.75 degrees, about 2degrees, about 2.5 degrees, about 3 degrees, or any values or rangesthere between (e.g., between about 0.1 degrees and about 1 degree). Withabout 1 degree of offset, the interface 5034 can be angled at about 44degrees or at about 46 degrees, depending on the direction of theoffset. The offset angle of the interface 5034 can angle the secondoutput beam 5038 even if the surface E is angled at 45 degrees relativeto the combined input beam. In some embodiments, the surface E canangled relative to the combined input beam by 45 degrees plus or minusan offset amount of about 0.02 degrees, about 0.05 degrees, about 0.1degrees, about 0.2 degrees, about 0.3 degrees, about 0.5 degrees, about0.75 degrees, about 1 degree, about 1.25 degrees, about 1.5 degrees,about 1.75 degrees, about 2 degrees, about 2.5 degrees, about 3 degrees,or any values or ranges there between (e.g., between about 0.1 degreesand about 1 degree). With about 1 degree of offset, the surface E can beangled at about 44 degrees or at about 46 degrees, depending on thedirection of the offset. The offset angle of the surface E can angle thefirst output beam 5036 even if the interface 5038 is angled at 45degrees relative to the combined input beam. In some embodiments, boththe interface 5038 and the surface E can be offset from 45 degrees. Insome embodiments, the surface E can be angled relative to the interface5034 by an angle of about 0.02 degrees, about 0.05 degrees, about 0.1degrees, about 0.2 degrees, about 0.3 degrees, about 0.5 degrees, about0.75 degrees, about 1 degree, about 1.25 degrees, about 1.5 degrees,about 1.75 degrees, about 2 degrees, about 2.5 degrees, about 3 degrees,or any values or ranges there between (e.g., between about 0.1 degreesand about 1 degree).

The interface 5028 and/or the surface I can have an angular offset in afirst direction (e.g., clockwise), and the interface 5038 and/or thesurface E can have an angular offset in a second direction (e.g.,counter clockwise) that is opposite the first direction. Various otherbase angles can be used instead of 45 degrees, and the surfaces and/orinterfaces can be offset from the base angle by different amounts ordirections so that the output beams are angled relative to each other.In some implementations, the output beams can be substantially parallelto each other (e.g., substantially parallel to the input beam), and thesizes of the prism elements and/or positions of the surfaces thatreflect and/or transmit the light can offset the parallel output beamsfrom each other. The interface 5028, the surface I, the surface E, andthe interface 5034 can be angled at 45 degrees, in some cases, althoughother angles can be used.

In some embodiments, the interface 5028 and the interface 5034 can beangled at 45 degrees, while the surface E and the surface I can have theoffset from 45 degrees. This design can be advantageous, in someimplementations, because the surface E and the surface I are exposed(e.g., to air) and are not coupled to other sides of other prismelements, which can facilitate polishing or fine tuning of the angles,etc.

With reference again to FIGS. 55 and 56 , the system 5000 can have abeam expander 5048. The beam expander 5048 can be positioned to receivethe first beam of light and the second beam of light output from theprism assembly 5025. The beam expander 5048 can be a cylindrical beamexpander configured to expand the first beam of light and the secondbeam of light along a first axis, while not expanding the first beam oflight and the second beam of light along a second axis that isorthogonal to the first axis. The beam expander 5048 can be a telescopicbeam expander, which can for example operate similar to a Galileantelescope (e.g., a cylindrical Galilean telescope). The beam expander5048 can have a first lens 5050 (e.g., a concave lens, a negativepowered lens, and/or a cylindrical lens) and a second lens 5052 (e.g., aconvex, a positive powered lens, and/or a cylindrical lens). The firstlens 5050 can be a doublet lens with two lens elements, or any number oflens elements, which can be configured to avoid, reduce, or correct foroptical aberration (e.g., spherical and/or chromatic), although anyother suitable lens configuration can be used. The convex lens 5052 canbe a doublet lens with two lens elements, or any number or lenselements, which can be configured to avoid, reduce, or correct foroptical aberration (e.g., spherical and/or chromatic), although anyother suitable lens configuration can be used. The focal point of thefirst lens 5050 can be at or on top of the focal point of the secondlens 5052. The beam expander 5048 can be afocal (e.g., where parallelrays of light into the first lens 5050 can produce parallel rays oflight out of the second lens 5052). The beam expander 5048 can use twoor more reflective surfaces (e.g., cylindrical reflective surfaces) toexpand the beams of light. In some embodiments, the beam expander 5048can be omitted. The amount of expansion provided by the beam expander5048 can affect the width of the output lines 5054 and 5056 (e.g., inthe Y direction).

The system 5000 can include a lens array 5013, which can be a microlensarray, and/or a cylindrical lens array. The lens array 5013 can have aplurality of cylindrical lens elements, which can be arranged in anarray. The lens array 5013 can use various features disclosed in the'348 application, for example. The lens array 5013 can be configured tomodify the distribution of light to smooth the intensity of light and/orto produce a substantially flat-top distribution of light for the outputlines. The first beam of light of the first wavelength (e.g., or centralaxis thereof) can intersect the lens array 5013 at a first location,which can be offset from a second location where the second beam oflight of the second wavelength (e.g., or central axis thereof) canintersect the lens array 5013. The first beam of light of the firstwavelength can be received at the lens array 5013 at a different anglethan the second beam of light of the second wavelength. In someembodiments, other optical elements can be used to modify thedistribution of the light (e.g., to produce a flat-top distribution oflight), such as a Powell lens, or an engineered diffuser, for example asdisclosed in the '348 application and/or the '213 patent. In someembodiments, the lens array 5013 can be omitted.

The system 5000 can include an objective lens system 5015, which can besimilar to other object lens systems disclosed in the '348 applicationand/or the '213 patent. Although the objective lens system 5015 is shownschematically in FIGS. 55 and 56 , it will be understood that theobjective lens system can include a number of lens elements and otherfeatures, such as an aperture or stop. The objective lens system 5015can be configured to receive the light (e.g., from the lens array 5013)and to output a first output line 5054 at the sample plane 5017 withlight of the first wavelength (e.g., Green) and a second output line5056 at the sample plane 5017 with light of the second wavelength (e.g.,Red), as shown for example in FIG. 59 . The objective lens system 5015can be a telecentric lens system. The telecentric lens system can beconfigured to output substantially parallel beams of light to producethe first output line 5054 and/or to produce the second output line5056. The objective lens system 5015 can have a stop or aperture, andthe first beam of light of the first wavelength can cross the secondbeam of light of the second wavelength at the objective stop oraperture.

FIG. 60 is a diagram illustrating an example of the beam profile of thefirst output line 5054 at the sample plane 5017 of the optical system ofFIGS. 55 and 56 . This beam has a thin line profile which can be usefulfor scanning certain types of samples such as in flow cells. FIG. 61 isa graph illustrating an example of the irradiance along the short-axisbeam profile (e.g., the Y-axis) at the sample plane 5017 of the systemof FIGS. 55 and 56 , with a peak around the 0.04 mm Y coordinate value.FIG. 62 is a graph illustrating an example of irradiance along thelong-axis beam profile (e.g., the X-axis) at the sample plane 5017 ofthe system of FIGS. 55 and 56 . Along the X-axis, the irradiance curveshows a sharp transition from near zero values to an approximatelyflat-top portion.

FIG. 63 is a diagram illustrating an example of the beam profile of thesecond output line 5056 at the sample plane 5017 of the optical systemof FIGS. 55 and 56 . This beam has a thin line profile which can beuseful for scanning certain types of samples such as in flow cells. FIG.64 a graph illustrating an example of the irradiance along theshort-axis beam profile (e.g., the Y-axis) at the sample plane 5017 ofthe system of FIGS. 55 and 56 , with a peak around the −0.04 mm Ycoordinate value. FIG. 65 is a graph illustrating an example ofirradiance along the long-axis beam profile (e.g., the X-axis) at thesample plane 5017 of the system of FIGS. 55 and 56 . Along the X-axis,the irradiance curve shows a sharp transition from near zero values toan approximately flat-top portion.

FIG. 66 is a side view of another example embodiment of a prism assembly5060. FIG. 67 is a perspective view of the example prism assembly 5060.The prism assembly 5060 of FIG. 66 can have four prism elements 5026a-d, with surfaces and other features similar to the prism assembly 5025embodiment of FIG. 57 , except as described herein. The fourth prismelement 5026 d can be a triangular prism with a corner cut off to make afour-side prism 5026 d. The prism assembly 5026 can be configured tooutput the first beam of light 5036 of the first wavelength (e.g.,Green) above the second beam of light 5038 of the second wavelength(e.g., Red). The resulting first output line 5054 of the firstwavelength of light (e.g., Green) can be below the resulting secondoutput line 5056 of the second wavelength of light (e.g., Red), such asthe opposite of the arrangement in FIG. 59 . The prism assembly 5060 ofFIG. 66 can cause the first light path of the first wavelength of lightto cross the second light path of the second wavelength of light insidethe prism assembly 5060, such as in the fourth prism element 5026 d. Inthe prism assembly 5025 of FIG. 57 , the first light path of the firstwavelength of light does not cross the second light path of the secondwavelength of light inside the prism assembly 5025.

FIG. 68 is a side view of another example embodiment of a prism assembly5065. In some embodiments, any of the prism elements 5026 a-d can bedivided into multiple prism sub-elements. For example, the second prism5026 b can include two prism sub-elements 5066 a and 5066 b, which canbe coupled to form a transition that transmits the first wavelength oflight. In some embodiments, the interface between the prism sub-elements5066 a and 5066 b can be index matched. The third prism 5026 c caninclude two prism sub-elements 5067 a and 5067 b, which can be coupledto form a transition that transmits the second wavelength of light. Insome embodiments, the interface between the prism sub-elements 5067 aand 5067 b can be index matched. One, two, or any number of the prismelements 5026 a-d can be divided into any number of prism sub-elements.In some embodiments, the prism element 5026 a and/or the prism element5026 d can be omitted.

FIG. 69 is a side view of another example embodiment of a prism assembly5070. The prism assembly 5070 can be similar to the prism assembly 5060of FIGS. 66 and 67 or to the prism assembly 5025 of FIG. 57 , exceptthat the first prism element 5026 a and the third prism element 5026 care combined into a single prism element 5068 a, and the second prismelement 5026 a and the fourth prism element 5026 d are combined into asingle prism element 5068 b. For the prism assembly 5070, the interface5030 and/or the interface 5032 shown in FIG. 57 can be eliminated, andthe first interface 5028 and the fourth interface 5034 shown in FIG. 57can be combined into a single interface 5035. The interface 5035 canhave a first interface portion 5035 a that is configured to transmit thefirst wavelength of light (e.g., Green) and to reflect the secondwavelength of light (e.g., Red), and a second interface portion 5035 bthat is configured to reflect the first wavelength of light (e.g.,Green) and to transmit the second wavelength of light (e.g., Red). Forexample, the first interface portion 5035 a can have a first polychroic(e.g., dichroic) coating or filter, and the second interface portion5035 b can have a second polychroic (e.g., dichroic) coating or filter.

FIG. 70 is a side view of an example embodiment of an illuminationsystem 5100. FIG. 71 is a top view of the example embodiment of theillumination system 5100. The illumination system 5100 can be similar tothe illumination system 5000 disclosed herein, except as discussedherein. The illumination system 5100 can produce three output lines ofthree different wavelengths of light. The illumination system 5100 canhave a collimating optical element 5111, which can be similar to thecollimating optical element 5011 disclosed herein. The illuminationsystem 5100 can have a prism assembly 5125, which can be similar to theother prism assemble embodiments disclosed herein. The prism assembly5125 can receive light having three different wavelengths of light, andthe prism assembly 5125 can split the light into three separate beams oflight of the three different wavelengths of light. Additionalinformation regarding the prism assembly 5125 is provided herein. Theillumination system 5100 can have a beam expander 5148 (e.g., acylindrical beam expander), which can be similar to the beam expander5048 disclosed herein. The beam expander 5148 can have a concave lens5150 and a convex lens 5152, which can be cylindrical, and which can besimilar to the lens 5050 and 5052 disclosed herein. The illuminationsystem 5100 can have a lens array 5113, which can be similar to the lensarray 5013 disclosed herein. The illumination system 5100 can have anobjective lens system 5115, which can be similar to the objective lenssystem 5015 disclosed herein. The illumination system 5100 canilluminate a sample plane 5117 similar to other embodiments disclosedherein.

FIG. 72 shows the first output line 5154, the second output line 5156,and the third output line 5157 produced by the system 5100. In theexample of FIG. 72 , the first output line 5154 can have light of afirst wavelength (e.g., 473 nm or Blue) 5151 b, the second output line5156 can have light of a second wavelength (e.g., 635 nm or Red) 5151 r,and the third output line 5157 can have light of a third wavelength(e.g., 525 nm or Green) 5151 g, although various different colors orwavelengths of light could be used.

FIG. 73 is a diagram illustrating an example of the beam profile of thefirst output line 5154 at the sample plane 5117 of the optical system ofFIGS. 70 and 71 . This beam has a thin line profile, which can be usefulfor scanning certain types of samples such as in flow cells. FIG. 74 isa graph illustrating an example of the irradiance along the short-axisbeam profile (e.g., the Y-axis) at the sample plane 5117 of the systemof FIGS. 70 and 71 , with a peak around the 0.07 mm Y coordinate value.FIG. 75 is a graph illustrating an example of irradiance along thelong-axis beam profile (e.g., the X-axis) at the sample plane 5117 ofthe system of FIGS. 70 and 71 . Along the X-axis, the irradiance curveshows a sharp transition from near zero values to an approximatelyflat-top portion.

FIG. 76 is a diagram illustrating an example of the beam profile of thesecond output line 5156 at the sample plane 5117 of the optical systemof FIGS. 70 and 71 . This beam has a thin line profile, which can beuseful for scanning certain types of samples such as in flow cells. FIG.77 is a graph illustrating an example of the irradiance along theshort-axis beam profile (e.g., the Y-axis) at the sample plane 5117 ofthe system of FIGS. 70 and 71 , with a peak around the −0.07 mm Ycoordinate value. FIG. 78 is a graph illustrating an example ofirradiance along the long-axis beam profile (e.g., the X-axis) at thesample plane 5117 of the system of FIGS. 70 and 71 . Along the X-axis,the irradiance curve shows a sharp transition from near zero values toan approximately flat-top portion.

FIG. 79 is a diagram illustrating an example of the beam profile of thethird output line 5157 at the sample plane 5117 of the optical system ofFIGS. 70 and 71 . This beam has a thin line profile, which can be usefulfor scanning certain types of samples such as in flow cells. FIG. 80 isa graph illustrating an example of the irradiance along the short-axisbeam profile (e.g., the Y-axis) at the sample plane 5117 of the systemof FIGS. 70 and 71 , with a peak around the 0.0 mm Y coordinate value.FIG. 81 is a graph illustrating an example of irradiance along thelong-axis beam profile (e.g., the X-axis) at the sample plane 5117 ofthe system of FIGS. 70 and 71 . Along the X-axis, the irradiance curveshows a sharp transition from near zero values to an approximatelyflat-top portion.

FIG. 82 shows an example embodiment of a prism assembly 5160, which canbe similar to the other prism assembly embodiments disclosed herein,except as described. The prism assembly 5160 can be configured to splita combined beam of light that has three different wavelengths of lightinto three beams of light that each include one of the three differentwavelengths. The prism assembly 5125 can have six prism elements 5126a-f. The first prism element 5126 a can be similar to the first prismelement 5026 a of the bi-color prism assembly 5025. The second prismelement 5126 b can be similar to the second prism element 5026 b of thebi-color prism assembly 5025. The third prism element 5126 c can besimilar to the third prism element 5026 c of the bi-color prism assembly5025. The fourth prism element 5126 d can be similar to the fourth prismelement 5026 d of the bi-color prism assembly 5025. The labeled sidesA-N from FIG. 57 can apply to the prism elements 5126 a-d, but are notreproduced in FIG. 82 .

The prism assembly 5125 can include a fifth prism element 5126 e, whichcan be between the first prism element 5126 a and the second prismelement 5126 b. The prism assembly 5125 can include a sixth prismelement 5126 f, which can be between the third prism element 5126 c andthe fourth prism element 2126 d. The fifth prism element 5126 e can havea first side O, a second side P, a third side Q, and a fourth side R.The fifth prism element 5126 e can be a 4-sided or rhomboid prism. Thesixth prism element 5126 f can have a first side S, a second side T, athird side U, and a fourth side V. The sixth or fifth prism element 5126e can be a 4-sided or rhomboid prism. The first side O of the fifthprism 5126 e can couple to the first prism 2126 a to form a firstinterface 5128, which can reflect a second wavelength of light 5151 r(e.g., red) while transmitting the first wavelength of light 5151 g(e.g., green) and the third wavelengths of light 5151 b (e.g., blue). Asecond interface 5130 between the first and third prism elements cantransmit the second wavelength of light 5151 r. A third interface 5132between the second prism element 5126 b and the fourth prism element5126 d can transmit the first wavelength of light 5151 g. A fourthinterface 5134 between the third side U of the sixth prism element 5126f and the fourth prism element 5126 d can be configured to reflect thefirst wavelength of light 5151 g, while transmitting the secondwavelength of light 5151 r and the third wavelength of light 5151 b. Afifth interface 5135 between the second surface P of the fifth prismelement 5126 e and the second prism element 5126 b can be configured totransmit the first wavelength of light 5151 g and to reflect the thirdwavelength of light 5151 b (e.g., toward the sixth prism element 5126f). A sixth interface 5137 between the third surface Q of the fifthprism element 5126 e and the first surface S of the sixth prism element5126 f can transmit light of the third wavelength 5151 b. A seventhinterface 5139 between the third prism assembly 5126 c and side T of thesixth prism assembly 5126 f can be configured to transmit the secondwavelength of light 5151 r while reflecting the third wavelength oflight 5151 b.

The interface 5135 can be at a 45 degree angle (e.g., relative to thecombined input beam), and the interface 5139 can be at a 45 degree angle(e.g., relative to the combined input beam), so that the third beam oflight 5141 of the third wavelength 5151 b (e.g., blue) that is output bythe prism assembly 5125 is substantially parallel to the combined inputbeam of light. The first beam 5136 of the first wavelength of light 5151r (e.g., red) and/or the second beam 5138 of the second wavelength oflight 5151 g (e.g., green) can be angled relative to each other and/orthe combined input beam, similar to the beams 5036 and 5038.

Any number of the prism element 5126 a-d can be divided into prismsub-elements, as discussed in connection with FIG. 68 . The fifth prismelement 5126 e can be integrated with the sixth prism element 5126 f,similar to the discussion for claim 69. The prism assembly 5125 can beconsidered a special case of the prism assembly 5060 of FIG. 66 , wherethe second prism element 5026 b is divided into two prism sub-elements5126 b and 5126 e, which can create a new interface 5135, which canreflect the third wavelength off light to split it off from the combinedbeam. Also, the third prism 5026 c can be divided into two prismsub-elements 5126 c and 5126 f, which can create a new interface 5139,which can reflect the third wavelength of light. In some embodiments,the prism 5126 a and/or the prism 5126 d can be omitted.

FIG. 83 is a side view of another example of a prism assembly 5180 thatis configured to produce three beams of light of different colors. FIG.84 is a perspective view of the prism assembly 5180. In the example ofFIG. 83 , the blue light beam 5151 b is above the green light beam 5151g, and the red light beam is below the green light beam 5151 r, with thered and blue light beams converging towards the green light beam. Thisarrangement can produce a red output line above a green output line, anda blue output line below the green output line. Various different colorsand arrangements are possible.

FIG. 85 is a side view of an example embodiment of an illuminationsystem 5200. FIG. 86 is a top view of the example embodiment of theillumination system 5200. The illumination system 5200 can be similar tothe illumination systems 5000 and 5100 disclosed herein, except asdiscussed herein. The system 5200 can include a first cylindrical lens5211 a, which can be configured to at least partially collimate thelight received from the optical fiber (or other light source). The firstcylindrical lens 5211 a can operate similar to the collimating lens 5011in one axis only (e.g., the X-axis). In some embodiments, a cylindricalreflector or other suitable optical element can be used for thecylindrical collimating optical element 5211 a. The system 5200 can havea second cylindrical collimating optical element 5211 b (e.g., acylindrical lens), which can be configured to at least partiallycollimate the light in another axis (e.g., the Y-axis). The secondoptical element 5211 b can be spaced apart from the first opticalelement 5211 a so that the light can expand further in one axis (e.g.,the Y-axis) compared to the other (e.g., the X-axis). Functionalitysimilar to the collimating optical element 5011 and the beam expander5048 of the system 5000 can be achieved with the optical elements 5211 aand 5211 b, using fewer components and less space. Because the beam isexpanded (e.g., in the Y-axis) before being delivered to the prismassembly 5225, the prism assembly 5225 of the system 5200 can be larger(e.g., at least in the Y-axis) than the prism assembly 5025. In somecases, using a beam expander 5048 after the prism assembly 5025 can beadvantageous to reduce the size of the prism assembly 5025, as comparedto the prism assembly 5225. The prism assembly 5225 can be configured toseparate light of a first wavelength 5251 r (e.g., red) from light of asecond wavelength 5251 g (e.g., green), similar to other embodimentsdisclosed herein, and some implementations can include other oradditional wavelengths of light and additional lines, as discussedherein. The system 5200 can include a lens array 5213, an objective lensor system 5215, and a sample plane 5217, which can be similar to thecorresponding components of the other systems disclosed herein.

In some cases, additional prism elements can be used to separateadditional wavelengths of light using the prism assembly. The system canbe configured to produce any number of output lines (e.g., 2 lines, 3lines, 4 lines, 5 lines, or more) of any suitable colors. In someembodiments the prism assembly can be omitted and other polychroicoptical elements can be used, such as polychroic (e.g., dichroic)plates. For example, with reference to FIG. 57 , dichroic plates can bearranged similar to the interface 5028 and the interface 5034, and/orreflective plates can be arranged similar to the surfaces E and I, whichcan output beams of light similar to the beams 5036 and 5038. Withreference to FIG. 82 , for three colors, polychroic plates can bearranged similar to the interface 5128, the interface 5135, theinterface 5139, and the interface 5134, and/or reflective plates can bearranged similar to surfaces E and I, which can output three beams oflight similar to the beams 5136, 5138, and 5141. In some cases, theprism assembly embodiments disclosed herein can be advantageous over asystem that uses polychroic to separate the different wavelengths oflight, because the prism assembly can have smaller optical path lengthsand can provide a more compact system. Systems using polychroic platesor mirrors can be beneficial for being easier to adjust relative anglesbetween the components, in some configurations.

The systems can include one or more features to reduce speckle. In someembodiments, a movably mirror (e.g., a MEMS mirror) can be used toreduce speckle, as discussed in the '348 application. The mirror can bedriven (e.g., vibrated or oscillated) to reduce speckle. In someembodiments, the system can have an optical fiber to provide light tothe system (e.g., from one or more lasers). The optical fiber can bemovable, and can be driven (e.g., vibrated) using a driver or actuatorto reduce speckle.

Additional Information

A wide variety of other variations, for example, to any of theembodiments or implementation disclosed herein, are possible. Componentscan be added, removed, and/or rearranged. Similarly, in any method orprocess disclosed herein, steps or operations can be added, removed,and/or rearranged. For example, although optical fiber is shown insystems including a microlens array such as a cylindrical lens array theoptical fiber can be included in any of the systems described hereinincluding systems that include different types of homogenizers includingsystems with diffusers, engineered diffusers, combination of diffusersand microlens arrays (e.g., cylindrical lens arrays), pairs ofcylindrical lens arrays, both those disclosed herein as well as insystems having other configurations and optical components. Also, asdiscussed above, the MEMS mirror can be scanned, for example along onedirection such as the direction where the cross-section of the beamorthogonal to the beam direction is the longest at the sample or sampleplane (such as in the y-direction) and/or may be scanned the fastestalong this direction and/or the most (e.g., largest distance or overlargest angle) in this direction. In some implementations, this onedirection is the only direction that the MEMS mirror tilts and/or scans.For example, MEMS mirrors that scan in one direction as opposed to twodirections may be configured to scan faster. In some implementations,the objective lens is disposed with respect to the microlens array orcylindrical lens array and the sample or sample plane is located withrespect to the objective lens such that the objective lens transformsangles of light exiting said microlens array or cylindrical lens arrayinto position along said sample or sample plane. For example, the sampleor sample plane may be the Fourier transform plane of the objective lensin some implementations. Other configurations are possible.

Configurations other than those described herein are possible. Thestructures, devices, systems, and methods may include additionalcomponents, features, and steps and any of these components, features,and steps may be excluded and may or may not be replaced with others.The arrangements may be different. Reference throughout thisspecification to “some embodiments,” “certain embodiments,” or “anembodiment” means that a particular feature, structure or characteristicdescribed in connection with the embodiment is included in at least someembodiments. Thus, appearances of the phrases “in some embodiments” or“in an embodiment” in various places throughout this specification arenot necessarily all referring to the same embodiment and may refer toone or more of the same or different embodiments. Furthermore, theparticular features, structures or characteristics may be combined inany suitable manner, as would be apparent to one of ordinary skill inthe art from this disclosure, in one or more embodiments.

As used in this application, the terms “comprising,” “including,”“having,” and the like are synonymous and are used inclusively, in anopen-ended fashion, and do not exclude additional elements, features,acts, operations, and so forth. Also, the term “or” is used in itsinclusive sense (and not in its exclusive sense) so that when used, forexample, to connect a list of elements, the term “or” means one, some,or all of the elements in the list.

Similarly, it should be appreciated that in the above description ofembodiments, various features are sometimes grouped together in a singleembodiment, figure, or description thereof for the purpose ofstreamlining the disclosure and aiding in the understanding of one ormore of the various inventive aspects. This method of disclosure,however, is not to be interpreted as reflecting an intention that anyclaim require more features than are expressly recited in that claim.Rather, inventive aspects lie in a combination of fewer than allfeatures of any single foregoing disclosed embodiment.

Some examples of some of certain embodiments disclosed and/or describedherein are listed below. These examples of various embodiments are notmeant to be limiting, but rather illustrate some of the embodiments ofsystems and methods that that are disclosed by this description andaccompanying figures. Such embodiments can include:

Although the inventions presented herein have been disclosed in thecontext of certain preferred embodiments and examples, it will beunderstood by those skilled in the art that the inventions extend beyondthe specifically disclosed embodiments to other alternative embodimentsand/or uses of the inventions and obvious modifications and equivalentsthereof. Thus, it is intended that the scope of the inventions hereindisclosed should not be limited by the particular embodiments describedabove.

1. An illumination system, comprising: a light source configured toprovide a combined beam of light having first light of a firstwavelength and second light of a second wavelength, wherein the firstlight and the second light are substantially coaxial; an opticalassembly comprising: a first surface configured to receive the combinedbeam of light and to transmit the first light of the first wavelengthand the second light of the second wavelength; a second surfaceconfigured to transmit the first light of the first wavelength along afirst optical path and to reflect the second light of the secondwavelength along a second optical path; a third surface configured toreflect the first light of the first wavelength, wherein the secondoptical path of the second light does not intersect the third surface; afourth surface configured to reflect the second light of the secondwavelength, wherein the first optical path of the first light does notintersect the fourth surface; a fifth surface configured to reflect thefirst light of the first wavelength and to transmit the second light ofthe second wavelength; and a sixth surface configured to output a firstbeam of light having the first light of the first wavelength from afirst location on the sixth surface, and to output a second beam oflight having the second light of the second wavelength from a secondlocation on the sixth surface that is offset from the first location,and wherein the first beam of light and the second beam of light have aconverging angle between the first beam and the second beam; acylindrical lens array positioned to receive the first beam of light andthe second beam of light, wherein the cylindrical lens array isconfigured to alter a distribution of light of the first beam to outputa first substantially flat-top distribution of light, and to alter adistribution of light of the second beam to output a secondsubstantially flat-top distribution of light; and an objective lenssystem positioned to receive the light from the cylindrical lens array,to output a first flat-top output line of the first wavelength at asample plane, and to output a second flat-top output line of the secondwavelength at the sample plane, wherein the second flat-top output lineis offset from the first flat-top output line.
 2. The illuminationsystem of claim 1, further comprising a beam expander between theoptical assembly and the cylindrical lens array.
 3. (canceled) 4.(canceled)
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 10. The illumination system of claim 1, wherein: the combinedbeam of light provided by the light source has third light of a thirdwavelength; the optical assembly comprises: a seventh surface configuredto transmit the first light of the first wavelength and to reflect thethird light of the third wavelength along a third optical path; and aneighth surface configured to transmit the second light of the secondwavelength and to reflect the third light of the third wavelength tooutput a third beam of light from a third location on the sixth surfacethat is offset from the first location and the second location; thecylindrical lens array is positioned to receive the third beam of light,wherein the cylindrical lens array is configured to alter a distributionof light of the third beam to output a third substantially flat-topdistribution of light; and the objective lens system is positioned toreceive the third light from the cylindrical lens array, to output athird flat-top output line of the third wavelength at the sample plane,wherein the third flat-top output line is offset from the first flat-topoutput line and from the second flat-top output line.
 11. (canceled) 12.(canceled)
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 15. The illumination system ofclaim 1, wherein the objective lens system comprises an objective stoppositioned so that the first beam of light and the second beam of lightcross at the objective stop.
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 18. Theillumination system of claim 1, wherein the second surface is angledrelative to the combined beam of light by 45 degrees, wherein the fifthsurface is angled relative to the combined beam of light by 45 degrees,wherein the third surface is angled relative to the combined beam oflight by a first angle that is not 45 degrees, and wherein the fourthsurface is angled relative to the combined beam of light by a secondangle that is not 45 degrees.
 19. The illumination system of claim 18,wherein the first angle differs from 45 degrees by about 0.1 degree toabout 1 degree, and wherein the second angle differs from 45 degrees byabout 0.1 degree to about 1 degree.
 20. The illumination system of claim1, wherein an angle between the first beam of light and the second beamof light is between about 1 mrad and about 30 mrad.
 21. The illuminationsystem of claim 1, wherein the optical assembly includes a firstpolychroic filter at the second surface that is configured to transmitthe first light of the first wavelength and to reflect the second lightof the second wavelength, or wherein the optical assembly includes asecond polychroic filter at the fifth surface that is configured toreflect the first light of the first wavelength and to transmit thesecond light of the second wavelength.
 22. (canceled)
 23. Theillumination system of claim 1, wherein the optical assembly is a prismassembly.
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 63. A prismassembly comprising: a first prism comprising: a first surfaceconfigured to receive a combined beam of light into the first prism, thecombined beam of light including a first wavelength of light and asecond wavelength of light; a second surface configured to receive thecombined beam of light; and a third surface; a second prism comprising:a first surface, wherein a first interface couples the second surface ofthe first prism to the first surface of the second prism, and whereinthe first interface is configured to transmit the first wavelength oflight into the second prism and to reflect the second wavelength oflight toward the third surface of the first prism; a second surfaceconfigured to reflect the first wavelength of light; and a third surfaceconfigured to receive the first wavelength of light reflected by thesecond surface of the second prism; a third prism comprising: a firstsurface, wherein a second interface couples the third surface of thefirst prism to the first surface of the third prism, and wherein thesecond interface is configured to transmit the second wavelength oflight into the third prism; a second surface configured to reflect thesecond wavelength of light; and a third surface configured to receivethe second wavelength of light reflected by the second surface of thethird prism; a fourth prism comprising: a first surface, wherein a thirdinterface couples the third surface of the second prism to the firstsurface of the fourth prism, and wherein the third interface isconfigured to transmit the first wavelength of light into the fourthprism; a second surface, wherein a fourth interface couples the thirdsurface of the third prism to the second surface of the fourth prism,and wherein the fourth interface is configured to reflect the firstwavelength of light and to transmit the second wavelength of light intothe fourth prism; and a third surface configured to output a first beamof the first wavelength of light and to output a second beam of thesecond wavelength of light.
 64. The prism assembly of claim 63, whereinthe second prism includes a fourth side extending between the first sideand the second side.
 65. The prism assembly of claim 63, wherein thethird prism includes a fourth side extending between the second side andthe third side.
 66. The prism assembly of claim 65, wherein the thirdprism includes two prism elements.
 67. The prism assembly of claim 63,wherein the second surface of the third prism is angled relative to thefirst interface by about 0.1 degree to about 1 degree, or wherein thesecond surface of the second prism is angled relative to the fourthinterface by about 0.1 degree to about 1 degree.
 68. (canceled)
 69. Theprism assembly of claim 63, comprising: a first polychroic filterpositioned at the first interface and configured to transmit the firstwavelength of light and to reflect the second wavelength of light; and asecond polychroic filter positioned at the fourth interface andconfigured to reflect the first wavelength of light and to transmit thesecond wavelength of light.
 70. An optical assembly, comprising: a firstsurface configured to: receive a combined beam of light having firstlight of a first wavelength and second light of a second wavelength;transmit the first light of the first wavelength along a first opticalpath; and reflect the second light of the second wavelength along asecond optical path; a second surface configured to reflect the firstlight of the first wavelength; a third surface configured to reflect thesecond light of the second wavelength; and a fourth surface configuredto: receive the first light of the first wavelength that was reflectedby the second surface; reflect the first light of the first wavelengthto produce a first beam of light having the first light of the firstwavelength; receive the second light of the second wavelength that wasreflected by the third surface; and transmit the second light of thesecond wavelength to produce a second beam of light having the secondlight of the second wavelength.
 71. The optical assembly of claim 70,wherein the optical assembly is configured to output the first beam oflight having the first light of the first wavelength from a firstlocation, and to output the second beam of light having the second lightof the second wavelength from a second location that is offset from thefirst location, and wherein the first beam of light and the second beamof light have a converging angle between the first beam and the secondbeam.
 72. The optical assembly of claim 70, wherein the second opticalpath of the second light does not intersect the second surface, andwherein the first optical path of the first light does not intersect thethird surface.
 73. The optical assembly of claim 70, comprising a lightsource configured to provide the combined beam of light having firstlight of a first wavelength and second light of a second wavelength,wherein the first light and the second light are substantially coaxial.74. (canceled)
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 78. Theoptical assembly of claim 70, comprising a cylindrical lens arraypositioned to receive the first beam of light and the second beam oflight, wherein the cylindrical lens array is configured to alter adistribution of light of the first beam to output a first substantiallyflat-top distribution of light, and to alter a distribution of light ofthe second beam to output a second substantially flat-top distributionof light.
 79. The optical assembly of claim 78, comprising an objectivelens system positioned to receive the light from the cylindrical lensarray, to output a first flat-top output line of the first wavelength ata sample plane, and to output a second flat-top output line of thesecond wavelength at the sample plane, wherein the second flat-topoutput line is offset from the first flat-top output line.
 80. Theoptical assembly of claim 79, wherein the objective lens systemcomprises an objective stop positioned so that the first beam of lightand the second beam of light cross at the objective stop.
 81. (canceled)82. The optical assembly of claim 70, wherein the third surface isangled relative to the first surface by about 0.1 degree to about 1degree, or wherein the second surface is angled relative to the fourthsurface by about 0.1 degree to about 1 degree.
 83. (canceled)
 84. Theoptical assembly of claim 70, comprising: a first polychroic filterpositioned at the first interface and configured to transmit the firstwavelength of light and to reflect the second wavelength of light; and asecond polychroic filter positioned at the fourth interface andconfigured to reflect the first wavelength of light and to transmit thesecond wavelength of light.
 85. The optical assembly of claim 70,wherein the optical assembly comprises a prism assembly or a pluralityof polychroic plates.
 86. (canceled)